Therapeutic regimen for hypertension

ABSTRACT

The invention relates to improved methods, devices, and kits for identifying and implementing an appropriate treatment regimen for subjects suffering from hypertension.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No. 15/314,641, filed Nov. 29, 2016, which is a U.S. National Stage Filing under 35 U.S.C. § 371 from International Application No. PCT/US2015/032651, filed May 27, 2015, and published as WO 2015/183938 on Dec. 3, 2015, which application claims benefit of the priority filing date of U.S. Provisional Application Ser. No. 62/004,460, filed May 29, 2014, the contents of which applications are specifically incorporated herein by reference in their entireties.

BACKGROUND

Hypertension (high blood pressure) is one of the most significant preventable contributors to disease and death in the world and represents the most common condition seen in the primary care setting (Kearney et al., Lancet 365:217-223 (2005)). According to the American Heart Association, approximately 78 million adults (1 in 3) living in the United States have hypertension with more than 5 million new diagnoses made each year. Of these individuals, 82% are aware they have it, 75% are currently being treated for it, but only 52% have their blood pressure under control (thus, ˜48% do not have adequate blood pressure control).

Hypertension can lead to myocardial infarction (heart attack), stroke, renal failure, and death if not detected early and treated appropriately. In 2009, high blood pressure was listed as a primary or contributing cause of death in about 350,000 of the approximate 2.4 million U.S. deaths (14% of all deaths). From 1999-2009 the number of deaths attributable to hypertension increased by 44%.

Refractory (or resistant) hypertension is defined as blood pressure that remains above clinical guideline goals in spite of concurrent use of three antihypertensive agents of different classes. Critically, refractory hypertension is noted in approximately 25-30% of all individuals being treated for hypertension. Refractory hypertension is a common clinical problem which contributes to the high levels of morbidity and mortality. In 2009, the direct and indirect economic burden on the United States health care system associated with hypertension was estimated at $51 billion.

Globally, nearly 1 billion individuals have been diagnosed with hypertension, with an estimate of an additional 400 million living with undiagnosed hypertension. Hypertension is the leading cause of premature death and the leading cause of cardiovascular disease worldwide. Similar to the continued upward trend in prevalence as seen in the United States, it is estimated that in 2025 about 1.56 billion adults will be living with hypertension. Because nearly two-thirds of the people living with hypertension worldwide reside in developing countries, providing optimal treatment at the lowest cost is critically important.

Unfortunately, despite a significant impulse in the medical community to move towards an “individualized medicine” approach to patient centered treatment, the current clinical treatment strategy is based on a set algorithm which does not take into account individual patient differences. Rather, physicians are guided to choose a drug (one out of many options) in a given class of drugs and use that specific drug as a “first line therapy” (typically initiating with the diuretic class) and titrate that specific drug of choice to therapeutic dosage regardless of efficacy. It is only after a prolonged course of treatment with that specific class of drug that clinical efficacy is determined (typically three months). At this stage, if clinical guideline goals for blood pressure have not been met, it is often recommended that the patient remain on the “first line therapy” whilst an additional drug from a different class of drugs (typically an Angiotensin converting enzyme inhibitor (ACE inhibitor) or Angiotensin II receptor blocker (ARB)) is added to the pharmacologic regimen. Again, this drug is titrated to recommended therapeutic dosage and another prolonged course of treatment is initiated before clinical efficacy is determined (an additional three months-six months since initiation of treatment). If at this point, clinical guideline goals for blood pressure have not been met, a third drug from a third class of drugs (typically a beta-blocker) is added and the process is repeated (another three months-nine months from initiation of treatment). Further, if clinical guideline goals have continued to be elusive, the diagnosis of refractory hypertension is added and the process is reinitiated with a different combination of drugs, different classes of drugs, different drug options within a given class of drugs, different dosages, or all of the above. Thus, from the time of initial diagnosis and the start of treatment to the point in which blood pressure is adequately controlled may take anywhere from three months to well over one year. This trial-and-error standard of care is clearly not optimal.

SUMMARY

The invention relates to improved methods, devices, and kits for identifying and implementing an appropriate treatment regimen for subjects suffering from hypertension. The methods, devices, and kits comprehensively assess common genetic variants in the cardiac, vascular, and renal systems in an effort to improve therapeutic guidance for high blood pressure treatment. Detection of an individual's genetic variants permits selection appropriate drug classes for that individual. Clinicians can then guide blood pressure therapy using knowledge that is specific to their individual patient, rather than the currently employed “trial-and-error” procedures that are based on population data and use of drugs with the least initial side effects.

One aspect of the invention is a method that includes:

-   -   (a) administering a loop diuretic to a subject as a first line         therapy, without a beta blocker and without a vasodilator, if         the subject's genome comprises a WNK1 nucleic acid with a         cytosine at the variable position of rs1159744 or rs2107614;     -   (b) administering a hydrochlorothiazide to a subject as a first         line therapy, without a beta blocker and without a vasodilator,         if the subject's genome comprises an ADD1 nucleic acid with a         thymine at the variable position of rs4961, or if the test         sample comprises a SLC12A3 nucleic acid with a thymine at the         variable position of rs1529927; or     -   (c) administering a hydrochlorothiazide to a subject as a first         line therapy, without a beta blocker and without a vasodilator,         if the subject's genome comprises an ADD1 nucleic acid with a         thymine at the variable position of rs4961, or if the test         sample comprises a SLC12A3 nucleic acid with a thymine at the         variable position of rs1529927.

Another aspect of the invention is a method that includes: administering a beta-blocker drug to a subject as a first line therapy, without a diuretic and without a hydrochlorothiazide, if the subject's genome does not comprise:

-   -   (a) a WNK1 nucleic acid with a cytosine at the variable position         of rs1159744;     -   (b) a WNK1 nucleic acid with a cytosine at the variable position         of rs2107614;     -   (c) an ADD1 nucleic acid with a thymine at the variable position         of rs4961; or     -   (d) a SLC12A3 nucleic acid with a thymine at the variable         position of rs1529927

but the subject's genome does comprise:

-   -   1. a CYP2D6 nucleic acid with an adenine at the variable         position of Rs3892097;     -   2. an ADRB1 nucleic acid with a cytosine at the variable         position of rs1801253;     -   3. an ADRB1 nucleic acid with an adenine at the variable         position of rs1801252;     -   4. an ADRB2 nucleic acid with a guanine at the variable position         of rs1042714; or     -   5. an ADRB2 nucleic acid with a guanine at the variable position         of rs1042713.

Another aspect of the invention is a method that includes: administering an angiotensin II receptor blocker to a subject as a first line therapy, without a diuretic, without a hydrochlorothiazide, and without a beta-blocker, if the subject's genome does not comprise:

-   -   (a) a WNK1 nucleic acid with a cytosine at the variable position         of rs1159744;     -   (b) a WNK1 nucleic acid with a cytosine at the variable position         of rs2107614;     -   (c) an ADD1 nucleic acid with a thymine at the variable position         of rs4961; or     -   (d) a SLC12A3 nucleic acid with a thymine at the variable         position of rs1529927

but the subject's genome does comprise:

-   -   1. a renin nucleic acid with a cytosine at the variable position         of rs12750834; or     -   2. an AGT1R nucleic acid with a cytosine at the variable         position of rs5186.

Another aspect of the invention is a method that includes: administering an ACE inhibitor to a subject without an angiotensin II receptor blocker as a first line therapy, without a diuretic, without a hydrochlorothiazide, and without a beta-blocker, if the subject's genome does not comprise:

-   -   (a) WNK1 nucleic acid with a cytosine at the variable position         of rs1159744;     -   (b) a WNK1 nucleic acid with a cytosine at the variable position         of rs2107614;     -   (c) an ADD1 nucleic acid with a thymine at the variable position         of rs4961; or     -   (d) a SLC12A3 nucleic acid with a thymine at the variable         position of rs1529927

but the subject's genome does comprise:

-   -   1. an ACE nucleic acid with a deletion in rs1799752; or     -   2. an AGT nucleic acid with a cytosine at the variable position         of rs699.

Another aspect of the invention is a method that includes: administering an amiloride as a first line therapy to a subject without an ACE inhibitor, without an angiotensin II receptor blocker, without a diuretic, without a hydrochlorothiazide, and without a beta-blocker, if the subject's genome does not comprise:

-   -   1. a WNK1 nucleic acid with a cytosine at the variable position         of rs1159744;     -   2. a WNK1 nucleic acid with a cytosine at the variable position         of rs2107614;     -   3. an ADD1 nucleic acid with a thymine at the variable position         of rs4961; or     -   4. a SLC12A3 nucleic acid with a thymine at the variable         position of rs1529927.

but if the subject's genome does comprise a SCNN1A nucleic acid with an adenine at the variable position of rs2228576.

The methods can also include administering a second line therapy drug after administration of the first line therapy for at least 1 month, wherein the second line therapy drug is selected from the group consisting of diuretic, a beta-blocker, an ACE inhibitor, a vasodilator, and a combination thereof.

Devices, compositions, methods, and kits are also described herein for identifying and implementing an appropriate treatment regimen for subjects suffering from hypertension.

DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic diagram illustrating the interplay between the heart, blood vessels, and kidney in blood pressure regulation.

FIG. 2 is a schematic diagram illustrating of the types of genes useful for evaluating hypertension, and representative single nucleotide polymorphisms that are correlated with blood pressure drug responses.

FIG. 3A-3B are schematic diagrams illustrating processing of test samples. For example, each subject can collect two swabs. The A swab can collect cell material from the inside of the right cheek, while the B swab can collect cell material from the left cheek. For FIG. 3A, the A swab can be the initial swab entered into the process (from DNA Extraction to Reporting). If the A swab fails, during DNA Yield and Purity Analysis, Genetic analysis, or the PCR QA Assay then the B swab can be entered into the system as illustrated. FIG. 3B shows the same process but without the steps of DNA Stocks Storage and Future Testing if the sample Passes Yield and Purity Assays.

FIG. 4 is a schematic diagram illustrating handling of DNA samples.

FIG. 5 is a schematic diagram illustrating processing of the sample after PCR amplification.

FIG. 6 illustrates alignment of sample results to a human reference sequence using the CLC DNA workbench program for creating an alignment file from which the allele call is made and added to the final SNP call report (SEQ ID NOs:82-85).

FIG. 7 is an example of 2% agarose gel used to score the presence or absence of a 288 bp ALU by visually examining the gel for either the higher molecular weight band (indicating the presence of the 288 bp ALU), the lower molecular weight band (indicating the absence of the 288 bp ALU), or both (indicating a heterozygous state).

FIG. 8 is a bar graph of urinary sodium output as a function of genetic variation of SCNN1A.

FIG. 9 is a bar graph of mean arterial blood pressure as a function of genetic variation of SCNN1A.

DETAILED DESCRIPTION

Methods, devices, and kits are described herein for selecting individualized hypertension treatment regimens that are more effective than currently available regimens. The methods, devices, and kits include assays for identifying genetic variants in individual subjects that make the individual more or less responsive to specific medications. When the appropriate medication is administered, the subject's blood pressure is appropriately controlled, and side effects are avoided. Genetic variants present in genes such as ADRB1, ADRB2, CYP2D6, angiotensin converting enzyme (ACE), angiotensinogen, angiotensin receptor, renin, Na⁺ channels (such as SCNN1A), adducin, sodium (Na⁺) chloride (Cl⁻) co-transporters (such as SLC12A3), and/or WNK1 genes are correlated with heightened or reduced responsiveness to various blood pressure medications. Although there are a number of hypertension drugs available on the market, subjects react differently to such drugs. The kits, methods, and devices described herein are useful for detecting which subjects benefit from which therapeutic regimen.

High Blood Pressure (Hypertension)

The development of high blood pressure in humans is the result of one or more of three physiologic maladaptations: 1) elevated cardiac output (liters of blood ejected from the heart per minute) that increases the amount of blood pressing against the vessels, 2) relatively narrow blood vessels that results in increased pressure towards the lumen of the blood vessel due to Poiseuille's Law (which describes the inverse relationship between the diameter of a tube (vessel) and the pressure against the walls of the tube (vessel), all else being equal), or 3) increased sodium (Na⁺) absorption in the kidney which results in increased blood volume (and the amount of blood pumped per minute, cardiac output) and subsequently increased outward pressure against the tubes (vessels).

Blood pressure is highly regulated and tightly controlled in humans, such that in the event of a drop in cardiac output, the heart sends a signal to the kidneys (via the proteins atrial natriuretic peptide and brain-type natriuretic peptide, among others) and vessels with the ultimate function of increasing Na⁺ reabsorption to increase plasma volume and vasoconstriction to increase blood pressure. Similarly, if there is a drop in blood pressure, there is an increase in cardiac output (primarily via an immediate increase in heart rate through inhibition of the parasympathetic nervous system) to compensate and an increase in the renin-angiotensin aldosterone system which results in both constriction of blood vessels (which increases blood pressure) and an increase in Na⁺ and, therefore, fluid retention in the kidney, which increases plasma volume and can increase blood pressure. Hence, the human body provides redundant functions to maintain blood pressure both in the short-term and in the long-term, by regulating the interplay between the heart, blood vessels, and kidney. FIG. 1 is a schematic diagram illustrating the interplay between the heart, blood vessels, and kidneys involved in regulating blood pressure.

Blood pressure therapy following diagnosis traditionally follows a regimented algorithm based on therapies effective across the general population. Currently, clinicians start a patient who has been diagnosed with high blood pressure on a diuretic (to reduce renal Na⁺ reabsorption). If such a diuretic does not work within a period of time, the clinician next tries a vasodilator, and if this is not effective, then a clinician will lastly prescribe a beta-blocker. This trial-and-error process to lower a patient's blood pressure can take several months, is costly, and threatens the health of the patient because the patient's hypertension is frequently not adequately controlled in a timely manner.

Such currently available methods are in stark contrast to the methods, devices, and kits described herein that involve specifically testing an individual's genetic profile and, as illustrated herein, basing therapeutic treatment on the results of such testing. Hence, the methods, devices, and kits described herein for evaluating a blood pressure genetic panel to improve treatment of hypertensive subjects, by quickly identifying more effective medications, thereby avoiding side effects and delays in treatment. Applicants' methods are therefore an improvement over the currently available trial-and-error procedures that frequently result in side effects and delays in effective treatment.

Ranking of Genotypes that Affect Therapy

The genotype of a subject can significantly affect the response of the subject to blood pressure medications because certain functional polymorphisms have greater effects on the physiology of a subject than others. The following is a summary of polymorphisms in order of their impact on blood pressure and which drugs should be employed by subjects with such genetic variations.

-   -   1. Subjects with the WNK1 polymorphism defined by rs1159744 (SEQ         ID NO:34, with cytosine at the variable position), benefit more         from loop diuretics.     -   2. Subjects with the WNK1 polymorphism defined by rs2107614 (SEQ         ID NO:33, with cytosine at the variable position), benefit more         from loop diuretics.     -   3. Subjects with the ADD1 polymorphism defined by rs4961 (SEQ ID         NO:27, with thymine at the variable position), benefit more from         hydrochlorothiazides.     -   4. Subjects with the SLC12A3 polymorphism defined by rs1529927         (SEQ ID NO:30, with thymine at the variable position), benefit         more from hydrochlorothiazides.         Any homozygous combination at one or more of the WNK1 Rs1159744,         WNK1 Rs2107614, ADD1 Rs4961, and SLC12A3 rs1529927 polymorphisms         means that diuretics should be the first-line therapy,         especially if the patient is heterozygous for polymorphisms in         genes responsive to beta-blockers or vasodilators.     -   5. Subjects with the CYP2D6 polymorphism defined by Rs3892097         (SEQ ID NO:10, with adenine at the variable position), plus the         ADRB1 polymorphism defined by rs1801253 (SEQ ID NO:3, with         cytosine as the variable nucleotide), plus the ADRB1         polymorphism defined by rs1801252 (SEQ ID NO:2, with adenine as         the variable nucleotide) benefit from beta-blockade classes of         drugs.     -   6. Subjects with the renin polymorphism defined by rs12750834         (SEQ ID NO:20, with cytosine as the variable nucleotide) plus         the AGT1R polymorphism defined by rs5186 (SEQ ID NO:16, with         cytosine as the variable nucleotide), which affect responses to         angiotensin II receptor blockers.     -   7. Subjects with the ACE deletion defined by Rs1799752 (SEQ ID         NO:35) and the AGT polymorphism defined by rs699 (SEQ ID NO: 14,         with cytosine as the variable nucleotide) can benefit from the         combination of an angiotensin II receptor blocker and an ACE         inhibitor.     -   8. Subjects with the SCNN1A polymorphism defined by rs2228576         (SEQ ID NO:22, with adenine as the variable position) can         benefit from administration of amiloride.     -   9. Subjects with the ADRB2 polymorphism defined by rs1042714         (SEQ ID NO:7, with guanine as the variable nucleotide) can         benefit from administration of a non-selective beta-blockade.     -   10. Subjects with the ADRB2 polymorphism defined by s1042713         (SEQ ID NO:6, with guanine as the variable nucleotide) can         benefit from administration of a non-selective beta-blockade.         All patients do not respond to same. Some subjects have         genotypes that can significantly affect their response to         medications. When clinicians employ currently available         procedures (diuretic first, then vasodilator, then beta         blocker), some patients will benefit but others will not respond         or will respond negatively. Hence, some patients would benefit         from initial administration of a vasodilator or a beta-blocker,         rather than a diuretic.         Beta-Blocker Responsive Polypeptides and Nucleic Acids

There are two primary receptors within the heart that influence both heart rate (chronotropic effect) and heart contractility (inotrpic effect) (Brodde, Am J Cardiol 62:24C-29C (1988), the beta-1 adrenergic receptors (β₁AR, encoded by the ADRB1 gene) and the beta-2 adrenergic receptors (β₂AR, encoded by the ADRB2 gene).

The heart is primary comprised of beta-1 adrenergic receptors, which are located on 80% of the ventricular wall surface, 70% of the atrial wall surface, and 95% of the sino-atrial (SA) node. The atria of the heart receive blood that returns from the body (right atria) of lungs (left atria) whereas the ventricles pump blood to the lungs (right ventricle) and body (left ventricle). The sino-atrial node primarily controls heart rate.

Although heart rate and cardiac contractility are primarily regulated by β₁AR, the β₂AR also play a role, primarily in cardiac contractility. Stimulation of either β₁AR or β₂AR can influence heart rate and cardiac contractility through increases in intracellular c-AMP and protein kinase A (PKA) which, ultimately, alter Ca⁺-channel sensitivity and reduce the threshold needed for an action potential. Therefore, cardiac output (and blood pressure) can be increased through increases in β₁AR and/or β₂AR activities. If a variant β₁AR or β₂AR gene encodes a more functional receptor, cardiac output is increased.

β₁AR and β₂AR activities can be modulated through the use of selective (e.g., atenolol and metoprolol) and non-selective (e.g., propranolol and carvedilol) beta-blockers. The selective beta-blockers are selective for inhibiting the β₁AR. The non-selective beta-blockers inhibit both β₁AR and β₂AR. Both types of beta-blockers tend to decrease blood pressure through a decrease in heart rate and cardiac contractility, which ultimately results in a decrease in cardiac output. Similarly, the administration of a β₂AR-agonist (e.g., albuterol sulfate) tends to increase cardiac output and heart rate (Snyder et al., Pharmacotherapy 31:748-756 (2011)). Thus, both β₁AR and β₂AR are important in the regulation of cardiac output.

Just as stimulation of these receptors can elevate cardiac output and increase blood pressure, so too can genetic variation of the genes that encode β₁AR and β₂AR (ADRB1 and ADRB2) elevate receptor activity and increase blood pressure. Conversely, some ADRB1 and ADRB2 genetic variants encode receptors with reduced activity. In addition, some ADRB1 and ADRB2 genetic variants exhibit reduced, or enhanced, responsiveness to blood pressure medications such as beta-blockers. Not all individuals respond similarly to beta-blockade, despite similar clinical and environmental conditions. As described herein, the effectiveness of beta-blockers is dependent to some extent upon the genetic make-up of the subjects to which the beta-blockers are administered.

Sequences for various adrenergic receptors are available, for example, from the National Center for Biotechnology Information (see website at ncbi.nlm.nih.gov).

For example, a full length human ADRB1 cDNA nucleotide sequence is available from the database maintained by the National Center for Biotechnology Information (see website at ncbi.nlm.nih.gov), which has accession number NM_00064 (GI:110349783), and is shown below as SEQ ID NO: 1.

1 GCACCACGCC GCCCGGGCTT CTGGGGTGTT CCCCAACCAC 41 GGCCCAGCCC TGCCACACCC CCCGCCCCCG GCCTCCGCAG 81 CTCGGCATGG GCGCGGGGGT GCTCGTCCTG GGCGCCTCCG 121 AGCCCGGTAA CCTGTCGTCG GCCGCACCGC TCCCCGACGG 161 CGCGGCCACC GCGGCGCGGC TGCTGGTGCC CGCGTCGCCG 201 CCCGCCTCGT TGCTGCCTCC CGCCAGCGAA A GCCCCGAGC 241 CGCTGTCTCA GCAGTGGACA GCGGGCATGG GTCTGCTGAT 281 GGCGCTCATC GTGCTGCTCA TCGTGGCGGG CAATGTGCTG 321 GTGATCGTGG CCATCGCCAA GACGCCGCGG CTGCAGACGC 361 TCACCAACCT CTTCATCATG TCCCTGGCCA GCGCCGACCT 401 GGTCATGGGG CTGCTGGTGG TGCCGTTCGG GGCCACCATC 441 GTGGTGTGGG GCCGCTGGGA GTACGGCTCC TTCTTCTGCG 481 AGCTGTGGAC CTCAGTGGAC GTGCTGTGCG TGACGGCCAG 521 CATCGAGACC CTGTGTGTCA TTGCCCTGGA CCGCTACCTC 561 GCCATCACCT CGCCCTTCCG CTACCAGAGC CTGCTGACGC 601 GCGCGCGGGC GCGGGGCCTC GTGTGCACCG TGTGGGCCAT 641 CTCGGCCCTG GTGTCCTTCC TGCCCATCCT CATGCACTGG 681 TGGCGGGCGG AGAGCGACGA GGCGCGCCGC TGCTACAACG 721 ACCCCAAGTG CTGCGACTTC GTCACCAACC GGGCCTACGC 761 CATCGCCTCG TCCGTAGTCT CCTTCTACGT GCCCCTGTGC 801 ATCATGGCCT TCGTGTACCT GCGGGTGTTC CGCGAGGCCC 841 AGAAGCAGGT GAAGAAGATC GACAGCTGCG AGCGCCGTTT 881 CCTCGGCGGC CCAGCGCGGC CGCCCTCGCC CTCGCCCTCG 921 CCCGTCCCCG CGCCCGCGCC GCCGCCCGGA CCCCCGCGCC 961 CCGCCGCCGC CGCCGCCACC GCCCCGCTGG CCAACGGGCG 1001 TGCGGGTAAG CGGCGGCCCT CGCGCCTCGT GGCCCTGCGC 1041 GAGCAGAAGG CGCTCAAGAC GCTGGGCATC ATCATGGGCG 1081 TCTTCACGCT CTGCTGGCTG CCCTTCTTCC TGGCCAACGT 1121 GGTGAAGGCC TTCCACCGCG AGCTGGTGCC CGACCGCCTC 1161 TTCGTCTTCT TCAACTGGCT GGGCTACGCC AACTCGGCCT 1201 TCAACCCCAT CATCTACTGC CGCAGCCCCG ACTTCCGCAA 1241 GGCCTTCCAG G GACTGCTCT GCTGCGCGCG CAGGGCTGCC 1281 CGCCGGCGCC ACGCGACCCA CGGAGACCGG CCGCGCGCCT 1321 CGGGCTGTCT GGCCCGGCCC GGACCCCCGC CATCGCCCGG 1361 GGCCGCCTCG GACGACGACG ACGACGATGT CGTCGGGGCC 1401 ACGCCGCCCG CGCGCCTGCT GGAGCCCTGG GCCGGCTGCA 1441 ACGGCGGGGC GGCGGCGGAC AGCGACTCGA GCCTGGACGA 1481 GCCGTGCCGC CCCGGCTTCG CCTCGGAATC CAAGGTGTAG 1521 GGCCCGGCGC GGGGCGCGGA CTCCGGGCAC GGCTTCCCAG 1561 GGGAACGAGG AGATCTGTGT TTACTTAAGA CCGATAGCAG 1601 GTGAACTCGA AGCCCACAAT CCTCGTCTGA ATCATCCGAG 1641 GCAAAGAGAA AAGCCACGGA CCGTTGCACA AAAAGGAAAG 1681 TTTGGGAAGG GATGGGAGAG TGGCTTGCTG ATGTTCCTTG 1721 TTGTTTTTTT TTTCTTTTCT TTTCTTTCTT CTTCTTTTTT 1741 TTTTTTTTTT TTTTTTCTGT TTGTGGTCCG GCCTTCTTTT 1801 GTGTGTGCGT GTGATGCATC TTTAGATTTT TTTCCCCCAC 1841 CAGGTGGTTT TTGACACTCT CTGAGAGGAC CGGAGTGGAA 1881 GATGGGTGGG TTAGGGGAAG GGAGAAGCAT TAGGAGGGGA 1921 TTAAAATCGA TCATCGTGGC TCCCATCCCT TTCCCGGGAA 1961 CAGGAACACA CTACCAGCCA GAGAGAGGAG AATGACAGTT 2001 TGTCAAGACA TATTTCCTTT TGCTTTCCAG AGAAATTTCA 2041 TTTTAATTTC TAAGTAATGA TTTCTGCTGT TATGAAAGCA 2081 AAGAGAAAGG ATGGAGGCAA AATAAAAAAA AATCACGTTT 2121 CAAGAAATGT TAAGCTCTTC TTGGAACAAG CCCCACCTTG 2161 CTTTCCTTGT GTAGGGCAAA CCCGCTGTCC CCCGCGCGCC 2201 TGGGTGGTCA GGCTGAGGGA TTTCTACCTC ACACTGTGCA 2241 TTTGCACAGC AGATAGAAAG ACTTGTTTAT ATTAAACAGC 2281 TTATTTATGT ATCAATATTA GTTGGAAGGA CCAGGCGCAG 2321 AGCCTCTCTC TGTGACATGT GACTCTGTCA ATTGAAGACA 2361 GGACATTAAA AGAGAGCGAG AGAGAGAAAC AGTTCAGATT 2401 ACTGCACATG TGGATAAAAA CAAAAACAAA AAAAAGGAGT 2441 GGTTCAAAAT GCCATTTTTG CACAGTGTTA GGAATTACAA 2481 AATCCACAGA AGATGTTACT TGCACAAAAA GAAATTAAAT 2521 ATTTTTTAAA GGGAGAGGGG CTGGGCAGAT CTTAAATAAA 2561 ATTCAAACTC TACTTCTGTT GTCTAGTATG TTATTGAGCT 2601 AATGATTCAT TGGGAAAATA CCTTTTTATA CTCCTTTATC 2641 ATGGTACTGT AACTGTATCC ATATTATAAA TATAATTATC 2681 TTAAGGATTT TTTATTTTTT TTTATGTCCA AGTGCCCACG 2721 TGAATTTGCT GGTGAAAGTT AGCACTTGTG TGTAAATTCT 2761 ACTTCCTCTT GTGTGTTTTA CCAAGTATTT ATACTCTGGT 2801 GCAACTAACT ACTGTGTGAG GAATTGGTCC ATGTGCAATA 2841 AATACCAATG AAGCACAATC AA

The rs1801252 single nucleotide polymorphism (SNP) is present in the ADRB1 gene, where the variable nucleotide at about position 231 (underlined) can be adenine in some individuals and guanine in others. The rs1801252 sequence (SEQ ID NO:2) is shown below, where the underlined A/G is the SNP.

CTCGTTGCTGCCTCCCGCCAGCGAA [A/G] GCCCCGAGCCGCTGTCTCA GCAGTG.

The rs1801253 single nucleotide polymorphism (SNP) is also present in the ADRB1 gene, where the variable nucleotide at about position 1251 (underlined) can be guanine in some individuals and cytosine in others. The rs1801253 sequence (SEQ ID NO:3) is shown below, where the underlined C/G is the SNP.

CCCCGACTTCCGCAAGGCCTTCCAG [C/G] GACTGCTCTGCTGCGCGCG CAGGGC.

The β₁AR polypeptide encoded by the ADRB1 cDNA with SEQ ID NO: 1 has the following sequence (SEQ ID NO:4).

1 MGAGVLVLGA SEPGNLSSAA PLPDGAATAA RLLVPASPPA 41 SLLPPASE S P EPLSQQWTAG MGLLMALIVL LIVAGNVLVI 81 VAIAKTPRLQ TLTNLFIMSL ASADLVMGLL VVPFGATIVV 121 WGRWEYGSFF CELWTSVDVL CVTASIETLC VIALDRYLAI 161 TSPFRYQSLL TRARARGLVC TVWAISALVS FLPILMHWWR 201 AESDEARRCY NDPKCCDFVT NRAYAIASSV VSFYVPLCIM 241 AFVYLRVFRE AQKQVKKIDS CERRFLGGPA RPPSPSPSPV 281 PAPAPPPGPP RPAAAAATAP LANGRAGKRR PSRLVALREQ 321 KALKTLGIIM GVFTLCWLPF FLANVVKAFH RELVPDRLFV 361 FFNWLGYANS AFNPIIYCRS PDFRKAFQ G L LCCARRAARR 401 RHATHGDRPR ASGCLARPGP PPSPGAASDD DDDDVVGATP 441 PARLLEPWAG CNGGAAADSD SSLDEPCRPG FASESKV

Note that the underlined amino acid at position 49 is serine because some individuals have SEQ ID NO: 1 or 2, where the variable nucleotide at about position 231 of SEQ ID NO: 1 is adenine. However, position 49 of SEQ ID NO:4 can be glycine in some individuals because those individual have guanine at nucleotide position 231 in SEQ ID NO:1.

Note also that the glycine at position 389 is an arginine (instead of glycine) as shown for SEQ ID NO:4 when position 1251 of SEQ ID NO: 1 is a cytosine.

Individuals with serine at β₁AR amino acid position 49 and/or arginine at position 389 are more responsive to beta-blockers than those with glycines at these positions. Hence, for example, an individual who expresses the β₁AR polypeptide with SEQ ID NO:4, will be more responsive to beta-blockers than an individual who expresses the β₁AR polypeptide with glycines at both positions 49 and 389.

A full length human ADRB2 cDNA nucleotide sequence is available from the database maintained by the National Center for Biotechnology Information (see website at ncbi.nlm.nih.gov), which has accession number NM_000024 (GI:283483994), and is shown below as SEQ ID NO:5.

1 GCACATAACG GGCAGAACGC ACTGCGAAGC GGCTTCTTCA 41 GAGCACGGGC TGGAACTGGC AGGCACCGCG AGCCCCTAGC 81 ACCCGACAAG CTGAGTGTGC AGGACGAGTC CCCACCACAC 121 CCACACCACA GCCGCTGAAT GAGGCTTCCA GGCGTCCGCT 161 CGCGGCCCGC AGAGCCCCGC CGTGGGTCCG CCCGCTGAGG 201 CGCCCCCAGC CAGTGCGCTC ACCTGCCAGA CTGCGCGCCA 241 TGGGGCAACC CGGGAACGGC AGCGCCTTCT TGCTGGCACC 281 CAAT A GAAGC CATGCGCCGG ACCACGACGT CACGCAG C AA 321 AGGGACGAGG TGTGGGTGGT GGGCATGGGC ATCGTCATGT 361 CTCTCATCGT CCTGGCCATC GTGTTTGGCA ATGTGCTGGT 401 CATCACAGCC ATTGCCAAGT TCGAGCGTCT GCAGACGGTC 441 ACCAACTACT TCATCACTTC ACTGGCCTGT GCTGATCTGG 481 TCATGGGCCT GGCAGTGGTG CCCTTTGGGG CCGCCCATAT 521 TCTTATGAAA ATGTGGACTT TTGGCAACTT CTGGTGCGAG 561 TTTTGGACTT CCATTGATGT GCTGTGCGTC ACGGCCAGCA 601 TTGAGACCCT GTGCGTGATC GCAGTGGATC GCTACTTTGC 641 CATTACTTCA CCTTTCAAGT ACCAGAGCCT GCTGACCAAG 681 AATAAGGCCC GGGTGATCAT TCTGATGGTG TGGATTGTGT 721 CAGGCCTTAC CTCCTTCTTG CCCATTCAGA TGCACTGGTA 761 CCGGGCCACC CACCAGGAAG CCATCAACTG CTATGCCAAT 801 GAGACCTGCT GTGACTTCTT CACGAACCAA GCCTATGCCA 841 TTGCCTCTTC CATCGTGTCC TTCTACGTTC CCCTGGTGAT 881 CATGGTCTTC GTCTACTCCA GGGTCTTTCA GGAGGCCAAA 921 AGGCAGCTCC AGAAGATTGA CAAATCTGAG GGCCGCTTCC 961 ATGTCCAGAA CCTTAGCCAG GTGGAGCAGG ATGGGCGGAC 1001 GGGGCATGGA CTCCGCAGAT CTTCCAAGTT CTGCTTGAAG 1041 GAGCACAAAG CCCTCAAGAC GTTAGGCATC ATCATGGGCA 1081 CTTTCACCCT CTGCTGGCTG CCCTTCTTCA TCGTTAACAT 1121 TGTGCATGTG ATCCAGGATA ACCTCATCCG TAAGGAAGTT 1161 TACATCCTCC TAAATTGGAT AGGCTATGTC AATTCTGGTT 1201 TCAATCCCCT TATCTACTGC CGGAGCCCAG ATTTCAGGAT 1241 TGCCTTCCAG GAGCTTCTGT GCCTGCGCAG GTCTTCTTTG 1281 AAGGCCTATG GGAATGGCTA CTCCAGCAAC GGCAACACAG 1321 GGGAGCAGAG TGGATATCAC GTGGAACAGG AGAAAGAAAA 1361 TAAACTGCTG TGTGAAGACC TCCCAGGCAC GGAAGACTTT 1401 GTGGGCCATC AAGGTACTGT GCCTAGCGAT AACATTGATT 1441 CACAAGGGAG GAATTGTAGT ACAAATGACT CACTGCTGTA 1481 AAGCAGTTTT TCTACTTTTA AAGACCCCCC CCCCCAACAG 1521 AACACTAAAC AGACTATTTA ACTTGAGGGT AATAAACTTA 1561 GAATAAAATT GTAAAATTGT ATAGAGATAT GCAGAAGGAA 1601 GGGCATCCTT CTGCCTTTTT TATTTTTTTA AGCTGTAAAA 1641 AGAGAGAAAA CTTATTTGAG TGATTATTTG TTATTTGTAC 1681 AGTTCAGTTC CTCTTTGCAT GGAATTTGTA AGTTTATGTC 1721 TAAAGAGCTT TAGTCCTAGA GGACCTGAGT CTGCTATATT 1761 TTCATGACTT TTCCATGTAT CTACCTCACT ATTCAAGTAT 1801 TAGGGGTAAT ATATTGCTGC TGGTAATTTG TATCTGAAGG 1841 AGATTTTCCT TCCTACACCC TTGGACTTGA GGATTTTGAG 1881 TATCTCGGAC CTTTCAGCTG TGAACATGGA CTCTTCCCCC 1921 ACTCCTCTTA TTTGCTCACA CGGGGTATTT TAGGCAGGGA 1961 TTTGAGGAGC AGCTTCAGTT GTTTTCCCGA GCAAAGTCTA 2001 AAGTTTACAG TAAATAAATT GTTTGACCAT GCCTTCATTG 2041 CAAAAAAAAA AAAAAAAA

The rs1042713 single nucleotide polymorphism (SNP) is present in the ADRB2 gene, where the variable nucleotide at about position 285 (underlined) can be in adenine some individuals and guanine in others. The rs1042713 sequence (SEQ ID NO:6) is shown below, where the underlined A/G is the SNP.

CAGCGCCTTCTTGCTGGCACCCAAT [A/G] GAAGCCATGCGCCGGACCA CGACGT.

The rs1042714 single nucleotide polymorphism (SNP) is also present in the ADRB2 gene, where the variable nucleotide at about position 318 (underlined) can be cytosine in some individuals and guanine in others. The rs1042714 sequence (SEQ ID NO:7) is shown below, where the underlined C/G is the SNP.

TGCGCCGGACCACGACGTCACGCAG [C/G] AAAGGGACGAGGTGTGGGT GGTGGG.

The β₂AR polypeptide encoded by the ADRB2 cDNA with SEQ ID NO:5 has the following sequence (SEQ ID NO:8).

1 MGQPGNGSAF LLAPN R SHAP DHDVTQ Q RDE VWVVGMGIVM 41 SLIVLAIVFG NVLVITAIAK FERLQTVTNY FITSLACADL 81 VMGLAVVPFG AAHILMKMWT FGNFWCEFWT SIDVLCVTAS 121 IETLCVIAVD RYFAITSPFK YQSLLTKNKA RVIILMVWIV 161 SGLTSFLPIQ MHWYRATHQE AINCYANETC CDFFTNQAYA 201 IASSIVSFYV PLVIMVFVYS RVFQEAKRQL QKIDKSEGRF 241 HVQNLSQVEQ DGRTGHGLRR SSKFCLKEHK ALKTLGIIMG 281 TFTLCWLPFF IVNIVHVIQD NLIRKEVYIL LNWIGYVNSG 321 FNPLIYCRSP DFRIAFQELL CLRRSSLKAY GNGYSSNGNT 361 GEQSGYHVEQ EKENKLLCED LPGTEDFVGH QGTVPSDNID 401 SQGRNCSTND SLL

Note that the underlined arginine at position 16 of SEQ ID NO:8 is arginine because some individuals have nucleotide sequence SEQ ID NO:5, where the nucleotide at about position 285 is adenine. However, position 16 of SEQ ID NO:8 can be glycine in some individuals because those individuals have guanine at nucleotide position 285 in SEQ ID NO:5.

Note also that the glutamine at position 27 of SEQ ID NO:8 is a glutamic acid when position 318 of nucleotide sequence SEQ ID NO:5 is a guanine.

Individuals with glycine at position 16 and/or glutamic acid at β₂AR position 27 are more responsive to beta-blockers than those with arginine and glutamine, respectively, at these positions. Hence, for example, an individual who expresses the β₂AR polypeptide with SEQ ID NO:5, will be more responsive to beta-blockers than an individual who expresses the β₂AR polypeptide with arginine and glutamine at positions 16 and 27.

The gene that encodes cytochrome P450 2D6 (CYP2D6) has been shown to alter the metabolism of the drugs in the beta-blocker class. This alteration in drug metabolism can alter the amount of bioavailable drug. Poor drug metabolizers tend to have more drugs available in the body for longer and will, therefore, have a greater response to therapy. In contrast, active metabolizers of a drug will have less of the drug available in their system and will respond poorly to therapy.

Because of the importance of CYP2D6 on beta-blocker metabolism, this gene is a useful marker of responsive to beta-blocker therapy.

A full length human CYP2D6 cDNA nucleotide sequence is available from the database maintained by the National Center for Biotechnology Information (see website at ncbi.nlm.nih.gov), which has accession number NM_000106.5 (GI:392513720), and is shown below as SEQ ID NO:9.

1 GTGCTGAGAG TGTCCTGCCT GGTCCTCTGT GCCTGGTGGG 41 GTGGGGGTGC CAGGTGTGTC CAGAGGAGCC CATTTGGTAG 81 TGAGGCAGGT ATGGGGCTAG AAGCACTGGT GCCCCTGGCC 121 GTGATAGTGG CCATCTTCCT GCTCCTGGTG GACCTGATGC 161 ACCGGCGCCA ACGCTGGGCT GCACGCTACC CACCAGGCCC 201 CCTGCCACTG CCCGGGCTGG GCAACCTGCT GCATGTGGAC 241 TTCCAGAACA CACCATACTG CTTCGACCAG TTGCGGCGCC 281 GCTTCGGGGA CGTGTTCAGC CTGCAGCTGG CCTGGACGCC 321 GGTGGTCGTG CTCAATGGGC TGGCGGCCGT GCGCGAGGCG 361 CTGGTGACCC ACGGCGAGGA CACCGCCGAC CGCCCGCCTG 401 TGCCCATCAC CCAGATCCTG GGTTTCGGGC CGCGTTCCCA 441 AGGGGTGTTC CTGGCGCGCT ATGGGCCCGC GTGGCGCGAG 481 CAGAGGCGCT TCTCCGTGTC CACCTTGCGC AACTTGGGCC 521 TGGGCAAGAA GTCGCTGGAG CAGTGGGTGA CCGAGGAGGC 561 CGCCTGCCTT TGTGCCGCCT TCGCCAACCA CTCC G GACGC 601 CCCTTTCGCC CCAACGGTCT CTTGGACAAA GCCGTGAGCA 641 ACGTGATCGC CTCCCTCACC TGCGGGCGCC GCTTCGAGTA 681 CGACGACCCT CGCTTCCTCA GGCTGCTGGA CCTAGCTCAG 721 GAGGGACTGA AGGAGGAGTC GGGCTTTCTG CGCGAGGTGC 761 TGAATGCTGT CCCCGTCCTC CTGCATATCC CAGCGCTGGC 801 TGGCAAGGTC CTACGCTTCC AAAAGGCTTT CCTGACCCAG 841 CTGGATGAGC TGCTAACTGA GCACAGGATG ACCTGGGACC 881 CAGCCCAGCC CCCCCGAGAC CTGACTGAGG CCTTCCTGGC 921 AGAGATGGAG AAGGCCAAGG GGAACCCTGA GAGCAGCTTC 961 AATGATGAGA ACCTGCGCAT AGTGGTGGCT GACCTGTTCT 1001 CTGCCGGGAT GGTGACCACC TCGACCACGC TGGCCTGGGG 1041 CCTCCTGCTC ATGATCCTAC ATCCGGATGT GCAGCGCCGT 1081 GTCCAACAGG AGATCGACGA CGTGATAGGG CAGGTGCGGC 1121 GACCAGAGAT GGGTGACCAG GCTCACATGC CCTACACCAC 1161 TGCCGTGATT CATGAGGTGC AGCGCTTTGG GGACATCGTC 1201 CCCCTGGGTG TGACCCATAT GACATCCCGT GACATCGAAG 1241 TACAGGGCTT CCGCATCCCT AAGGGAACGA CACTCATCAC 1281 CAACCTGTCA TCGGTGCTGA AGGATGAGGC CGTCTGGGAG 1321 AAGCCCTTCC GCTTCCACCC CGAACACTTC CTGGATGCCC 1361 AGGGCCACTT TGTGAAGCCG GAGGCCTTCC TGCCTTTCTC 1401 AGCAGGCCGC CGTGCATGCC TCGGGGAGCC CCTGGCCCGC 1441 ATGGAGCTCT TCCTCTTCTT CACCTCCCTG CTGCAGCACT 1481 TCAGCTTCTC GGTGCCCACT GGACAGCCCC GGCCCAGCCA 1521 CCATGGTGTC TTTGCTTTCC TGGTGAGCCC ATCCCCCTAT 1561 GAGCTTTGTG CTGTGCCCCG CTAGAATGGG GTACCTAGTC 1601 CCCAGCCTGC TCCCTAGCCA GAGGCTCTAA TGTACAATAA 1641 AGCAATGTGG TAGTTCCAAA AAAAAAAAAA AAA

The rs3892097 single nucleotide polymorphism (SNP) is present in the CYP2D6 gene, where the variable nucleotide at a splice site at about position 595 in SEQ ID NO:9 (underlined), which can be in adenine some individuals and guanine in others.

The rs3892097 sequence (SEQ ID NO: 10) of the CYP2D6 gene is shown below, where the underlined A/G is the SNP.

CCCTTACCCGCATCTCCCACCCCCA [A/G] GACGCCCCTTTCGCCCCAAC GGTCT. Because the SNP occurs near a splice site, the sequences to the left of the SNP site in SEQ ID NO: 10 do not appear in the SEQ ID NO:9 nucleotide CYP2D6 cDNA sequence.

The cytochrome P450 2D6 polypeptide encoded by the CYP2D6 cDNA with SEQ ID NO:9 has the following sequence (SEQ ID NO: 11).

1 MGLEALVPLA VIVAIFLLLV DLMHRRQRWA ARYPPGPLPL 41 PGLGNLLHVD FQNTPYCFDQ LRRRFGDVFS LQLAWTPVVV 81 LNGLAAVREA LVTHGEDTAD RPPVPITQIL GFGPRSQGVF 121 LARYGPAWRE QRRFSVSTLR NLGLGKKSLE QWVTEEAACL 161 CAAFANHS G R PFRPNGLLDK AVSNVIASLT CGRRFEYDDP 201 RFLRLLDLAQ EGLKEESGFL REVLNAVPVL LHIPALAGKV 241 LRFQKAFLTQ LDELLTEHRM TWDPAQPPRD LTEAFLAEME 281 KAKGNPESSF NDENLRIVVA DLFSAGMVTT STTLAWGLLL 321 MILHPDVQRR VQQEIDDVIG QVRRPEMGDQ AHMPYTTAVI 361 HEVQRFGDIV PLGVTHMTSR DIEVQGFRIP KGTTLITNLS 401 SVLKDEAVWE KPFRFHPEHF LDAQGHFVKP EAFLPFSAGR 441 RACLGEPLAR MELFLFFTSL LQHFSFSVPT GQPRPSHHGV 481 FAFLVSPSPY ELCAVPR

Note that the underlined glycine at position 169 of SEQ ID NO: 11 is glycine because some individuals have nucleotide sequence SEQ ID NO:9, where the nucleotide at about position 595 is guanine. However, position 169 of SEQ ID NO:11 can be arginine in some individuals because those individuals have adenine at nucleotide position 295 in SEQ ID NO:9.

A patient with that is homozygous for adenine (AA) at the rs3892097 variable site will express CYP2D6 with arginine at position 169 and will not metabolize metoprolol and propranolol as quickly as those with guanine (glycine). Hence, homozygous individuals with adenine (AA) at the rs3892097 variable site have higher plasma levels of metoprolol and propranolol after taking these drugs than subjects that are not homozygous for adenine (AA) at the rs3892097 variable site. Homozygous individuals with adenine (AA) at the rs3892097 variable site would respond more normally to atenolol and carvedilol, which do not require CYP2D6 for their metabolism.

Vasodilation Therapy

Dilation of blood vessels results in decreases in blood pressure, whereas constriction of blood vessels results in increases in blood pressure. The blood vessels are controlled through local neural signaling that is largely under parasympathetic control, and circulating hormones that are largely under sympathetic control, as well as other circulating proteins. Blood pressure increases following stimulation of the angiotensin receptors, which results in vasoconstriction. Angiotensin receptors are stimulated by angiotensin II, which is converted from angiotensin I through the angiotensin converting enzyme (ACE). Angiotensin II is a potent vasoconstrictor and actively inhibits bradykinin, which is a potent vasodilator.

Therefore, angiotensin converting enzyme is a common target of blood pressure therapy. ACE inhibitors such as lisinopril promote vasodilation which, ultimately, reduces the bioavailability of angiotensin-II. Similarly, angiotensin II receptor antagonists such as losartan act as competitive inhibitors, which decrease the number of receptors that are available to bind to angiotensin-II. Despite the method used for promoting vasodilation (through reductions in ACE or receptor inhibition) the end result, on average in the population, is vasodilation which results in a drop in blood pressure due to the inverse relationship between the size of the vessel and the pressure exerted on the vessel, all else being equal. Despite this benefit there is a “bell-curve” response to these therapies in humans. Not all individuals are responsive to vasodilator therapy.

Several functional polymorphisms of the genes that encode for ACE and angiotensin-II receptors exist, which can affect how a subject responds to vasodilation.

Examples of functional ACE polymorphisms include the insertion or deletion polymorphisms such as a 287 base pair fragment (Ulgen et al., Coron Artery Dis 18:153-157 (2007)). The rs1799752 polymorphism is an insertion/deletion in an intron of the ACE gene, and with the sequence (SEQ ID NO: 12) shown below, where sequences in the bracket are the insertion/deletion.

TCCCATTTCTCTAGACCTGCTGCCT[-/ATACAGTCACTTTTTTTTTTTT TTTGAGACGGAGTCTCGCTCTGTCGCCC]ATACAGTCACTTTTATGTGGT TTCG. The deletion removes the bracketed nucleic acid segment so that the rs1799752 polymorphism will have the following sequence (SEQ ID NO:35).

TCCCATTTCTCTAGACCTGCTGCCTATACAGTCACTTTTATGTGGTTTCG.

Research indicates that such an ACE deletion polymorphism results in higher ACE plasma levels and greater reduction in ejection fraction in patients following myocardial infraction (likely from elevations in blood pressure) (McNamara et al., J Am Coll Cardiol 44:2019-2026 (2004); Pilati et al., Congest Heart Fail 10:87-93 (2004). In addition, patients with the deletion polymorphism are more likely to have left-ventricular hypertrophy when compared to patients with the insertion polymorphism (left-ventricular hypertrophy results secondary to prolonged exposure to high blood pressure). Subjects with the deletion polymorphism would therefore be most responsive to ACE-inhibition or angiotensin-II receptor inhibition.

At least one functional variant of angiotensin has been found in humans: a cytosine to threonine substitution at nucleotide 4072 (Pilbrow et al., Hypertension 49:322-327 (2007); Tang et al., Am Heart J 143:854-860 (2002)). Human angiotensinogen is expressed from the AGT gene. A cDNA nucleotide sequence for human angiotensinogen is provided below as SEQ ID NO: 13 (accession number NM_000029.3 GI:188595658, from the NCBI database).

1 ATCCCATGAG CGGGCAGCAG GGTCAGAAGT GGCCCCCGTG 41 TTGCCTAAGC AAGACTCTCC CCTGCCCTCT GCCCTCTGCA 81 CCTCCGGCCT GCATGTCCCT GTGGCCTCTT GGGGGTACAT 121 CTCCCGGGGC TGGGTCAGAA GGCCTGGGTG GTTGGCCTCA 161 GGCTGTCACA CACCTAGGGA GATGCTCCCG TTTCTGGGAA 201 CCTTGGCCCC GACTCCTGCA AACTTCGGTA AATGTGTAAC 241 TCGACCCTGC ACCGGCTCAC TCTGTTCAGC AGTGAAACTC 281 TGCATCGATC ACTAAGACTT CCTGGAAGAG GTCCCAGCGT 321 GAGTGTCGCT TCTGGCATCT GTCCTTCTGG CCAGCCTGTG 361 GTCTGGCCAA GTGATGTAAC CCTCCTCTCC AGCCTGTGCA 401 CAGGCAGCCT GGGAACAGCT CCATCCCCAC CCCTCAGCTA 441 TAAATAGGGC ATCGTGACCC GGCCGGGGGA AGAAGCTGCC 481 GTTGTTCTGG GTACTACAGC AGAAGGGTAT GCGGAAGCGA 521 GCACCCCAGT CTGAGATGGC TCCTGCCGGT GTGAGCCTGA 561 GGGCCACCAT CCTCTGCCTC CTGGCCTGGG CTGGCCTGGC 601 TGCAGGTGAC CGGGTGTACA TACACCCCTT CCACCTCGTC 641 ATCCACAATG AGAGTACCTG TGAGCAGCTG GCAAAGGCCA 681 ATGCCGGGAA GCCCAAAGAC CCCACCTTCA TACCTGCTCC 721 AATTCAGGCC AAGACATCCC CTGTGGATGA AAAGGCCCTA 761 CAGGACCAGC TGGTGCTAGT CGCTGCAAAA CTTGACACCG 801 AAGACAAGTT GAGGGCCGCA ATGGTCGGGA TGCTGGCCAA 841 CTTCTTGGGC TTCCGTATAT ATGGCATGCA CAGTGAGCTA 881 TGGGGCGTGG TCCATGGGGC CACCGTCCTC TCCCCAACGG 921 CTGTCTTTGG CACCCTGGCC TCTCTCTATC TGGGAGCCTT 961 GGACCACACA GCTGACAGGC TACAGGCAAT CCTGGGTGTT 1001 CCTTGGAAGG ACAAGAACTG CACCTCCCGG CTGGATGCGC 1041 ACAAGGTCCT GTCTGCCCTG CAGGCTGTAC AGGGCCTGCT 1081 AGTGGCCCAG GGCAGGGCTG ATAGCCAGGC CCAGCTGCTG 1121 CTGTCCACGG TGGTGGGCGT GTTCACAGCC CCAGGCCTGC 1161 ACCTGAAGCA GCCGTTTGTG CAGGGCCTGG CTCTCTATAC 1201 CCCTGTGGTC CTCCCACGCT CTCTGGACTT CACAGAACTG 1241 GATGTTGCTG CTGAGAAGAT TGACAGGTTC ATGCAGGCTG 1281 TGACAGGATG GAAGACTGGC TGCTCCCTGA T GGGAGCCAG 1321 TGTGGACAGC ACCCTGGCTT TCAACACCTA CGTCCACTTC 1361 CAAGGGAAGA TGAAGGGCTT CTCCCTGCTG GCCGAGCCCC 1401 AGGAGTTCTG GGTGGACAAC AGCACCTCAG TGTCTGTTCC 1441 CATGCTCTCT GGCATGGGCA CCTTCCAGCA CTGGAGTGAC 1481 ATCCAGGACA ACTTCTCGGT GACTCAAGTG CCCTTCACTG 1521 AGAGCGCCTG CCTGCTGCTG ATCCAGCCTC ACTATGCCTC 1561 TGACCTGGAC AAGGTGGAGG GTCTCACTTT CCAGCAAAAC 1601 TCCCTCAACT GGATGAAGAA ACTATCTCCC CGGACCATCC 1641 ACCTGACCAT GCCCCAACTG GTGCTGCAAG GATCTTATGA 1681 CCTGCAGGAC CTGCTCGCCC AGGCTGAGCT GCCCGCCATT 1721 CTGCACACCG AGCTGAACCT GCAAAAATTG AGCAATGACC 1761 GCATCAGGGT GGGGGAGGTG CTGAACAGCA TTTTTTTTGA 1801 GCTTGAAGCG GATGAGAGAG AGCCCACAGA GTCTACCCAA 1841 CAGCTTAACA AGCCTGAGGT CTTGGAGGTG ACCCTGAACC 1881 GCCCATTCCT GTTTGCTGTG TATGATCAAA GCGCCACTGC 1921 CCTGCACTTC CTGGGCCGCG TGGCCAACCC GCTGAGCACA 1961 GCATGAGGCC AGGGCCCCAG AACACAGTGC CTGGCAAGGC 2001 CTCTGCCCCT GGCCTTTGAG GCAAAGGCCA GCAGCAGATA 2041 ACAACCCCGG ACAAATCAGC GATGTGTCAC CCCCAGTCTC 2081 CCACCTTTTC TTCTAATGAG TCGACTTTGA GCTGGAAAGC 2121 AGCCGTTTCT CCTTGGTCTA AGTGTGCTGC ATGGAGTGAG 2161 CAGTAGAAGC CTGCAGCGGC ACAAATGCAC CTCCCAGTTT 2201 GCTGGGTTTA TTTTAGAGAA TGGGGGTGGG GAGGCAAGAA 2241 CCAGTGTTTA GCGCGGGACT ACTGTTCCAA AAAGAATTCC 2281 AACCGACCAG CTTGTTTGTG AAACAAAAAA GTGTTCCCTT 2321 TTCAAGTTGA GAACAAAAAT TGGGTTTTAA AATTAAAGTA 2361 TACATTTTTG CATTGCCTTC GGTTTGTATT TAGTGTCTTG 2401 AATGTAAGAA CATGACCTCC GTGTAGTGTC TGTAATACCT 2441 TAGTTTTTTC CACAGATGCT TGTGATTTTT GAACAATACG 2481 TGAAAGATGC AAGCACCTGA ATTTCTGTTT GAATGCGGAA 2521 CCATAGCTGG TTATTTCTCC CTTGTGTTAG TAATAAACGT 2561 CTTGCCACAA TAAGCCTCCA 2581 AAAAAAA

The rs699 single nucleotide polymorphism (SNP) is present in the AGT gene, where the variable nucleotide is at about position 1311 in SEQ ID NO: 13 (underlined), which can be in thymine some individuals and cytosine in others. The rs699 sequence (SEQ ID NO: 14) is shown below, where the underlined C/T is the SNP.

GGATGGAAGACTGGCTGCTCCCTGA [C/T] GGGAGCCAGTGTGGACAGCA CCCTG.

The human angiotensinogen polypeptide encoded by the AGT cDNA with SEQ ID NO:13 has the following sequence (SEQ ID NO:15).

1 MRKRAPQSEM APAGVSLRAT ILCLLAWAGL AAGDRVYIHP 41 FHLVIHNEST CEQLAKANAG KPKDPTFIPA PIQAKTSPVD 81 EKALQDQLVL VAAKLDTEDK LRAAMVGMLA NFLGFRIYGM 121 HSELWGVVHG ATVLSPTAVF GTLASLYLGA LDHTADRLQA 161 ILGVPWKDKN CTSRLDAHKV LSALQAVQGL LVAQGRADSQ 201 AQLLLSTVVG VFTAPGLHLK QPFVQGLALY TPVVLPRSLD 241 FTELDVAAEK IDRFMQAVTG WKTGCSL M GA SVDSTLAFNT 281 YVHFQGKMKG FSLLAEPQEF WVDNSTSVSV PMLSGMGTFQ 321 HWSDIQDNFS VTQVPFTESA CLLLIQPHYA SDLDKVEGLT 361 FQQNSLNWMK KLSPRTIHLT MPQLVLQGSY DLQDLLAQAE 401 LPAILHTELN LQKLSNDRIR VGEVLNSIFF ELEADEREPT 441 ESTQQLNKPE VLEVTLNRPF LFAVYDQSAT ALHFLGRVAN 481 PLSTA

Note that the underlined methionine at position 268 of SEQ ID NO: 15 is methionine because some individuals have nucleotide sequence SEQ ID NO: 13, where the nucleotide at about position 1311 is thymine. However, position 268 of SEQ ID NO: 15 can be threonine in some individuals because those individuals have cytosine at nucleotide position 1311 in SEQ ID NO: 13.

The threonine polymorphism of angiotensin results in higher angiotensin levels and higher resting blood pressure values. Therefore, patients with the threonine genetic variant will benefit primarily from an ACE inhibitor (preventing the conversion of the higher levels of angiotensin I to angiotensin II) or an angiotensin receptor inhibitor.

An example of a functional polymorphism of an angiotensin II receptor type-1 involves an adenine to cytosine substitution at nucleotide 1166 (Miller et al. Kidney Int 56:2173-2180 (1999); Baudin, Pharmacogenomics 3:65-73 (2002)). Human angiotensin II receptor type-1 is expressed from the AGT1R gene. One example of an AGT1R single nucleotide polymorphism is the so-called A1166→C polymorphism, which is in the 3′ untranslated region of the AGT1R gene. This A1166→C polymorphism is also identified as the rs5186 single nucleotide polymorphism (SNP), which has the following sequence (SEQ ID NO: 16) where the underlined A/C is the variable SNP site.

TGCAGCACTTCACTACCAAATGAGC [A/C] TTAGCTACTTTTCAGAATTG AAGGA. A portion of a 3′ untranslated region of the AGT1R gene with NCBI accession number NG_008468.1 (GI:198041751) is shown below (SEQ ID NO:17) that contains the rs5186 SNP with the variant nucleotide (adenine) identified below in bold and with underlining.

48961 ATTCAACTAG GCATCATACG TGACTGTAGA ATTGCAGATA 49001 TTGTGGACAC GGCCATGCCT ATCACCATTT GTATAGCTTA 49041 TTTTAACAAT TGCCTGAATC CTCTTTTTTA TGGCTTTCTG 49081 GGGAAAAAAT TTAAAAGATA TTTTCTCCAG CTTCTAAAAT 49121 ATATTCCCCC AAAAGCCAAA TCCCACTCAA ACCTTTCAAC 49181 AAAAATGAGC ACGCTTTCCT ACCGCCCCTC AGATAATGTA 49201 AGCTCATCCA CCAAGAAGCC TGCACCATGT TTTGAGGTTG 49241 AGTGACATGT TCGAAACCTG TCCATAAAGT AATTTTGTGA 49301 AAGAAGGAGC AAGAGAACAT TCCTCTGCAG CACTTCACTA 49321 CCAAATGAGC A TTAGCTACT TTTCAGAATT GAAGGAGAAA 49361 ATGCATTATG TGGACTGAAC CGACTTTTCT AAAGCTCTGA 49401 ACAAAAGCTT TTCTTTCCTT TTGCAACAAG ACAAAGCAAA 49441 GCCACATTTT GCATTAGACA GATGACGGCT GCTCGAAGAA 49481 CAATGTCAGA AACTCGATGA ATGTGTTGAT TTGAGAAATT 49521 TTACTGACAG AAATGCAATC TCCCTAGCCT GCTTTTGTCC 49561 TGTTATTTTT TATTTCCACA TAAAGGTATT TAGAATATAT 49601 TAAATCGTTA GAGGAGCAAC AGGAGATGAG AGTTCCAGAT 49641 TGTTCTGTCC AGTTTCCAAA GGGCAGTAAA GTTTTCGTGC

This polymorphism has been shown to influence resting blood pressure values which suggest which patients may benefit more from angiotensin-II receptor inhibition. Specifically, patients with the C variant of the angiotensin receptor type I tend to demonstrate higher resting blood pressure values, have more detrimental cardiovascular events, and have a greater chance of developing high blood pressure during pregnancy, when compared to the A variant. Subjects with the C variant will therefore be more responsive to angiotensin receptor blockers.

A cDNA sequence for human angiotensin II receptor is provided in the NCBI database as accession number X65699.1 (GI:510983), which has the following sequence (SEQ ID NO:18).

1 GGCAGCAGCG AGTGACAGGA CGTCTGGACC GGCGCGCCGC 41 TAGCAGCTCT GCCGGGCCGC GGCGGTGATC GATGGGAGCG 81 GCTGGAGCGG ACCCAGCGAG TGAGGGCGCA CAGCCGGACG 121 CCGAGGCGGC GGGCGGGAGA CCGCACCGCG ACGCCGGCCC 161 TCGGCGGACG AGTCGAGCGC CCGGGCGCGG GTGTATTTGA 201 TATAGTGTTT GCAACAAATT CGACCCAGGT GATCAAAATG 241 ATTCTCAACT CTTCTACTGA AGATGGTATT AAAAGAATCC 281 AAGATGATTG TCCCAAAGCT GGAAGGCATA ATTACATATT 321 TGTCATGATT CCTACTTTAT ACAGTATCAT CTTTGTGGTG 361 GGAATATTTG GAAACAGCTT GGTGGTGATA GTCATTTACT 401 TTTATATGAA GCTGAAGACT GTGGCCAGTG TTTTTCTTTT 441 GAATTTAGCA CTGGCTGACT TATGCTTTTT ACTGACTTTG 481 CCACTATGGG CTGTCTACAC AGCTATGGAA TACCGCTGGC 521 CCTTTGGCAA TTACCTATGT AAGATTGCTT CAGCCAGCGT 561 CAGTTTCAAC CTGTACGCTA GTGTGTTTCT ACTCACGTGT 601 CTCAGCATTG ATCGATACCT GGCTATTGTT CACCCAATGA 641 AGTCCCGCCT TCGACGCACA ATGCTTGTAG CCAAAGTCAC 681 CTGCATCATC ATTTGGCTGC TGGCAGGCTT GGCCAGTTTG 721 CCAGCTATAA TCCATCGAAA TGTATTTTTC ATTGAGAACA 761 CCAATATTAC AGTTTGTGCT TTCCATTATG AGTCCCAAAA 801 TTCAACCCTC CCGATAGGGC TGGGCCTGAC CAAAAATATA 841 CTGGGTTTCC TGTTTCCTTT TCTGATCATT CTTACAAGTT 881 ATACTCTTAT TTGGAAGGCC CTAAAGAAGG CTTATGAAAT 921 TCAGAAGAAC AAACCAAGAA ATGATGATAT TTTTAAGATA 961 ATTATGGCAA TTGTGCTTTT CTTTTTCTTT TCCTGGATTC 1001 CCCACCAAAT ATTCACTTTT CTGGATGTAT TGATTCAACT 1041 AGGCATCATA CGTGACTGTA GAATTGCAGA TATTGTGGAC 1081 ACGGCCATGC CTATCACCAT TTGTATAGCT TATTTTAACA 1121 ATTGCCTGAA TCCTCTTTTT TATGGCTTTC TGGGGAAAAA 1161 ATTTAAAAGA TATTTTCTCC AGCTTCTAAA ATATATTCCC 1201 CCAAAAGCCA AATCCCACTC AAACCTTTCA ACAAAAATGA 1241 GCACGCTTTC CTACCGCCCC TCAGATAATG TAAGCTCATC 1281 CACCAAGAAG CCTGCACCAT GTTTTGAGGT TGAGTGACAT 1321 GTTCGAAACC TGTCCATAAA GTAATTTTGT GAAAGAAGGA 1361 GCAAGAGAAC ATTCCTCTGC AGCACTTCAC TACCAAATGA 1401 GC A TTAGCTA CTTTTCAGAA TTGAAGGAGA AAATGCATTA 1441 TGTGGACTGA ACCGACTTTT CTAAAGCTCT GAACAAAAGC 1481 TTTTCTTTCC TTTTGCAACA AGACAAAGCA AAGCCACATT 1521 TTGCATTAGA CAGATGACGG CTGCTCGAAG AACAATGTCA 1561 GAAACTCGAT GAATGTGTTG ATTTGAGAAA TTTTACTGAC 1601 AGAAATGCAA TCTCCCTAGC CTGCTTTTGT CCTGTTATTT 1641 TTTATTTCCA CATAAAGGTA TTTAGAATAT ATTAACTCGT 1681 TAGAGGAGCA ACAGGAGATG AGAGTTCCAG ATTGTTCTGT 1721 CCAGTTTCCA AAGGGCAGTA AAGTTTTCGT GCCTGTTTTC 1761 AGCTATTAGC AACTGTGCCT ACACTTGCAC CTGGTCTGCA 1801 CATTTTGTAC AAAGATATGC TTAAGCAGTA GTCGTCAAGT 1841 TGCAGATCTT TGTTGTGAAA TTCAACCTGT GTCTTATAGG 1881 TTTACACTGC CAAAACAATG CCCGTAAGAT GGCTTATTTG 1921 TATAATGGTG TTACCTAAAG TCACATATAA AAGTTAAACT 1961 ACTTGTAAAG GTGCTGCACT GGTCCCAAGT AGTAGTGTCT 2001 TCCTAGTATA TTAGTTTGAT TTAATATCTG AGAAGTGTAT 2041 ATAGTTTGTG GTAAAAAGAT TATATATCAT AAAGTATGCC 2081 TTCCTGTTTA AAAAAAGTAT ATATTCTACA CATATATGTA 2121 TATGTATATC TATATCTCTA AACTGCTGTT AATTGATTAA 2161 AATCTGGCAA AGTTATATTT ACCCC

In addition to angiotensin, angiotensin II receptors and ACE, renin has been shown to be a potent vasoconstrictor that can result in high blood pressure. Renin converts angiotensinogen to angiotensin I which can result in vasoconstriction due to the down-stream effects (angiotensin-I conversion to angiotensin II through ACE). One example of a functional and common renin polymorphism (Vangjeli et al., Circulation Cardiovascular genetics 3:53-59 (2010)) can influence the blood pressure response to vasodilator therapy. This renin polymorphism is present in rs12750834. The nucleotide sequence surrounding the renin rs12750834 single nucleotide polymorphism is shown below, where the underlined A/G in the sequence (SEQ ID NO: 19) is the SNP.

AGAACACCAAAGCAGGCTTAATCTG [A/G] GGGCACTTACAGAGACTGCT TTAAA. The complementary sequence of SEQ ID NO: 19 is the following sequence (SEQ ID NO:20).

TTTAAAGCAGTCTCTGTAAGTGCCC [C/T] CAGATTAAGCCTGCTTTGGT GTTCT. Note that the cytosine to thymine substitution is a guanine to adenine substitution in the opposite strand.

The rs12750834 SNP contains a cytosine to thymine substitution, or a guanine to adenine substitution depending upon the DNA strand, at about nucleotide position 5312 upstream of the renin start site. The cytosine (guanine) variant of renin has been shown to correlate with greater reduction in blood pressure upon administration of angiotensin II receptor blockers such as valsartan.

Sodium/Diuretic Regulation of Blood Pressure

The kidneys are the center of long-term blood pressure regulation. Alterations in Na⁺ reabsorption in the kidneys result in alterations in fluid retention, which leads to increases or decreases in blood plasma volume as well as to changes in the pressure against the vessels. There are several proteins that are important in renal Na⁺ handling and in the response to diuretic therapy including the epithelial Na⁺ channels, alpha-adducin, the Na⁺Cl⁻ co-transporter, and lysine deficient protein kinase-1 (WNK).

The epithelial sodium (Na⁺) channel is responsible for Na⁺ reabsorption on the apical portion of epithelial cells in the kidneys. The Na⁺ channel is made up of three different subunits: the alpha, beta, and gamma. The alpha subunit of the epithelial Na⁺ channel is highly functional and removal of this subunit abolishes channel activity in cell and animal models. The gamma subunit is also extremely important in channel function. Functional gamma genetic variants result in pseudohypoaldosteronism type-I and Liddle's syndrome, two severe genetic diseases resulting in salt wasting and high salt conservation (salt sensitivity), respectively. Adducin is made up of an alpha, beta, and gamma subunit. The alpha subunit increases sodium (Na⁺) reabsorption in the kidneys through the activity of Na⁺K⁺ ATPase (which moves Na⁺ and potassium into and out of cells). The sodium (Na⁺) chloride (Cl⁻) co-transporter is important in regulating Na⁺ and Cl⁻ movement between the kidney and the rest of the body. Active Na⁺—Cl⁻ transport results in Na⁺ reabsorption and can, therefore, result in higher blood pressure. The WNK1 protein is a key regulator of long-term Na⁺ and chloride Cl⁻ reabsorption in the kidneys. WNK1 regulates the activity of Na⁺—Cl⁻ co-transporters. If a patient has a more active WNK1 genotype, they likely have greater Na⁺ and Cl⁻ reabsorption in the kidneys which can increase blood volume and, therefore, pressure on the vessels.

A functional and common polymorphism of the gene that encodes the epithelial Na+channel (SCNN1A) has been identified, where the polymorphism is an alanine to threonine substitution at about position 663-722. A cDNA sequence for the human SCNN1A gene is available from the NCBI database as accession number NM_001159576.1 (GI:227430288). This sequence is provided below as SEQ ID NO:21.

1 AAACAGAAGG CAGATAGAGA GGGAGTGAGA GGCAGGAGCT 41 GAGACACAGA TCCTGGAGGA AGAAGACCAA AGGAAGGGGG 81 CAGAGACAGA AAGGGAGGTG CTAGGACAAA ACTCGAAAGG 121 TGGCCCTATC AGGGAAGCAG AGGAGAGGCC GTTCTAGGGA 161 AGCCCAGCTC CGGCACTTTT GGCCCCAACT CCCGCAGGTC 201 TGCTGGCTCC AGGAAAGGTG GAGGAGGGAG GGAGGAGTGG 241 GAGAATGTGG GCGCAGGGTG GGACATGGGC ATGGCCAGGG 281 GCAGCCTCAC TCGGGTTCCA GGGGTGATGG GAGAGGGCAC 321 TCAGGGCCCA GAGCTCAGCC TTGACCCTGA CCCTTGCTCT 361 CCCCAATCCA CTCCGGGGCT CATGAAGGGG AACAAGCTGG 401 AGGAGCAGGA CCCTAGACCT CTGCAGCCCA TACCAGGTCT 441 CATGGAGGGG AACAAGCTGG AGGAGCAGGA CTCTAGCCCT 481 CCACAGTCCA CTCCAGGGCT CATGAAGGGG AACAAGCGTG 521 AGGAGCAGGG GCTGGGCCCC GAACCTGCGG CGCCCCAGCA 561 GCCCACGGCG GAGGAGGAGG CCCTGATCGA GTTCCACCGC 601 TCCTACCGAG AGCTCTTCGA GTTCTTCTGC AACAACACCA 641 CCATCCACGG CGCCATCCGC CTGGTGTGCT CCCAGCACAA 681 CCGCATGAAG ACGGCCTTCT GGGCAGTGCT GTGGCTCTGC 721 ACCTTTGGCA TGATGTACTG GCAATTCGGC CTGCTTTTCG 761 GAGAGTACTT CAGCTACCCC GTCAGCCTCA ACATCAACCT 801 CAACTCGGAC AAGCTCGTCT TCCCCGCAGT GACCATCTGC 841 ACCCTCAATC CCTACAGGTA CCCGGAAATT AAAGAGGAGC 881 TGGAGGAGCT GGACCGCATC ACAGAGCAGA CGCTCTTTGA 921 CCTGTACAAA TACAGCTCCT TCACCACTCT CGTGGCCGGC 961 TCCCGCAGCC GTCGCGACCT GCGGGGGACT CTGCCGCACC 1001 CCTTGCAGCG CCTGAGGGTC CCGCCCCCGC CTCACGGGGC 1041 CCGTCGAGCC CGTAGCGTGG CCTCCAGCTT GCGGGACAAC 1081 AACCCCCAGG TGGACTGGAA GGACTGGAAG ATCGGCTTCC 1121 AGCTGTGCAA CCAGAACAAA TCGGACTGCT TCTACCAGAC 1161 ATACTCATCA GGGGTGGATG CGGTGAGGGA GTGGTACCGC 1201 TTCCACTACA TCAACATCCT GTCGAGGCTG CCAGAGACTC 1241 TGCCATCCCT GGAGGAGGAC ACGCTGGGCA ACTTCATCTT 1281 CGCCTGCCGC TTCAACCAGG TCTCCTGCAA CCAGGCGAAT 1321 TACTCTCACT TCCACCACCC GATGTATGGA AACTGCTATA 1361 CTTTCAATGA CAAGAACAAC TCCAACCTCT GGATGTCTTC 1401 CATGCCTGGA ATCAACAACG GTCTGTCCCT GATGCTGCGC 1441 GCAGAGCAGA ATGACTTCAT TCCCCTGCTG TCCACAGTGA 1481 CTGGGGCCCG GGTAATGGTG CACGGGCAGG ATGAACCTGC 1521 CTTTATGGAT GATGGTGGCT TTAACTTGCG GCCTGGCGTG 1561 GAGACCTCCA TCAGCATGAG GAAGGAAACC CTGGACAGAC 1601 TTGGGGGCGA TTATGGCGAC TGCACCAAGA ATGGCAGTGA 1641 TGTTCCTGTT GAGAACCTTT ACCCTTCAAA GTACACACAG 1681 CAGGTGTGTA TTCACTCCTG CTTCCAGGAG AGCATGATCA 1721 AGGAGTGTGG CTGTGCCTAC ATCTTCTATC CGCGGCCCCA 1761 GAACGTGGAG TACTGTGACT ACAGAAAGCA CAGTTCCTGG 1801 GGGTACTGCT ACTATAAGCT CCAGGTTGAC TTCTCCTCAG 1841 ACCACCTGGG CTGTTTCACC AAGTGCCGGA AGCCATGCAG 1881 CGTGACCAGC TACCAGCTCT CTGCTGGTTA CTCACGATGG 1921 CCCTCGGTGA CATCCCAGGA ATGGGTCTTC CAGATGCTAT 1961 CGCGACAGAA CAATTACACC GTCAACAACA AGAGAAATGG 2001 AGTGGCCAAA GTCAACATCT TCTTCAAGGA GCTGAACTAC 2041 AAAACCAATT CTGAGTCTCC CTCTGTCACG ATGGTCACCC 2081 TCCTGTCCAA CCTGGGCAGC CAGTGGAGCC TGTGGTTCGG 2121 CTCCTCGGTG TTGTCTGTGG TGGAGATGGC TGAGCTCGTC 2161 TTTGACCTGC TGGTCATCAT GTTCCTCATG CTGCTCCGAA 2201 GGTTCCGAAG CCGATACTGG TCTCCAGGCC GAGGGGGCAG 2241 GGGTGCTCAG GAGGTAGCCT CCACCCTGGC ATCCTCCCCT 2281 CCTTCCCACT TCTGCCCCCA CCCCATGTCT CTGTCCTTGT 2321 CCCAGCCAGG CCCTGCTCCC TCTCCAGCCT TGACAGCCCC 2361 TCCCCCTGCC TATGCCACCC TGGGCCCCCG CCCATCTCCA 2401 GGGGGCTCTG CAGGGGCCAG TTCCTCC A CC TGTCCTCTGG 2441 GGGGGCCCTG AGAGGGAAGG AGAGGTTTCT CACACCAAGG 2481 CAGATGCTCC TCTGGTGGGA GGGTGCTGGC CCTGGCAAGA 2521 TTGAAGGATG TGCAGGGCTT CCTCTCAGAG CCGCCCAAAC 2561 TGCCGTTGAT GTGTGGAGGG GAAGCAAGAT GGGTAAGGGC 2601 TCAGGAAGTT GCTCCAAGAA CAGTAGCTGA TGAAGCTGCC 2641 CAGAAGTGCC TTGGCTCCAG CCCTGTACCC CTTGGTACTG 2681 CCTCTGAACA CTCTGGTTTC CCCACCCAAC TGCGGCTAAG 2721 TCTCTTTTTC CCTTGGATCA GCCAAGCGAA ACTTGGAGCT 2761 TTGACAAGGA ACTTTCCTAA GAAACCGCTG ATAACCAGGA 2801 CAAAACACAA CCAAGGGTAC ACGCAGGCAT GCACGGGTTT 2841 CCTGCCCAGC GACGGCTTAA GCCAGCCCCC GACTGGCCTG 2881 GCCACACTGC TCTCCAGTAG CACAGATGTC TGCTCCTCCT 2921 CTTGAACTTG GGTGGGAAAC CCCACCCAAA AGCCCCCTTT 2961 GTTACTTAGG CAATTCCCCT TCCCTGACTC CCGAGGGCTA 3001 GGGCTAGAGC AGACCCGGGT AAGTAAAGGC AGACCCAGGG 3041 CTCCTCTAGC CTCATACCCG TGCCCTCACA GAGCCATGCC 3081 CCGGCACCTC TGCCCTGTGT CTTTCATACC TCTACATGTC 3121 TGCTTGAGAT ATTTCCTCAG CCTGAAAGTT TCCCCAACCA 3161 TCTGCCAGAG AACTCCTATG CATCCCTTAG AACCCTGCTC 3201 AGACACCATT ACTTTTGTGA ACGCTTCTGC CACATCTTGT 3241 CTTCCCCAAA ATTGATCACT CCGCCTTCTC CTGGGCTCCC 3281 GTAGCACACT ATAACATCTG CTGGAGTGTT GCTGTTGCAC 3321 CATACTTTCT TGTACATTTG TGTCTCCCTT CCCAACTAGA 3361 CTGTAAGTGC CTTGCGGTCA GGGACTGAAT CTTGCCCGTT 3401 TATGTATGCT CCATGTCTAG CCCATCATCC TGCTTGGAGC 3441 AAGTAGGCAG GAGCTCAATA AATGTTTGTT GCATGAAGGA 3481 AAAAAAAAAA AAAAAAA

The rs2228576 single nucleotide polymorphism (SNP) is present in the SCNN1A gene, where the variable nucleotide is at about position 2428 in SEQ ID NO:21 (underlined), which can be adenine in some individuals and guanine in others. The rs2228576 sequence (SEQ ID NO:22) is shown below, where the underlined A/G is the SNP.

GGGCTCTGCAGGGGCCAGTTCCTCC [A/G] CCTGTCCTCTGGGGGGGCCC TGAGA

The human the epithelial Na+channel encoded by the SCNN1A cDNA with SEQ ID NO:21 has the following sequence (SEQ ID NO:23).

1 MGMARGSLTR VPGVMGEGTQ GPELSLDPDP CSPQSTPGLM 41 KGNKLEEQDP RPLQPIPGLM EGNKLEEQDS SPPQSTPGLM 81 KGNKREEQGL GPEPAAPQQP TAEEEALIEF HRSYRELFEF 121 FCNNTTIHGA IRLVCSQHNR MKTAFWAVLW LCTFGMMYWQ 161 FGLLFGEYFS YPVSLNINLN SDKLVFPAVT ICTLNPYRYP 201 EIKEELEELD RITEQTLFDL YKYSSFTTLV AGSRSRRDLR 241 GTLPHPLQRL RVPPPPHGAR RARSVASSLR DNNPQVDWKD 281 WKIGFQLCNQ NKSDCFYQTY SSGVDAVREW YRFHYINILS 321 RLPETLPSLE EDTLGNFIFA CRFNQVSCNQ ANYSHFHHPM 361 YGNCYTFNDK NNSNLWMSSM PGINNGLSLM LRAEQNDFIP 401 LLSTVTGARV MVHGQDEPAF MDDGGFNLRP GVETSISMRK 441 ETLDRLGGDY GDCTKNGSDV PVENLYPSKY TQQVCIHSCF 481 QESMIKECGC AYIFYPRPQN VEYCDYRKHS SWGYCYYKLQ 521 VDFSSDHLGC FTKCRKPCSV TSYQLSAGYS RWPSVTSQEW 561 VFQMLSRQNN YTVNNKRNGV AKVNIFFKEL NYKTNSESPS 601 VTMVTLLSNL GSQWSLWFGS SVLSVVEMAE LVFDLLVIMF 641 LMLLRRFRSR YWSPGRGGRG AQEVASTLAS SPPSHFCPHP 681 MSLSLSQPGP APSPALTAPP PAYATLGPRP SPGGSAGASS 721 S T CPLGGP

Another cDNA sequence for the human SCNN1A gene with the same SNP is available from the NCBI database as accession number NM_001038.5 (GI:227430285). This sequence is provided below as SEQ ID NO:24.

1 CTTGCCTGTC TGCGTCTAAA GCCCCTGCCC AGAGTCCGCC 41 TTCTCAGGTC CAGTACTCCC AGTTCACCTG CCCTCGGGAG 81 CCCTCCTTCC TTCGGAAAAC TCCCGGCTCT GACTCCTCCT 121 CAGCCCCTCC CCCCGCCCTG CTCACCTTTA ATTGAGATGC 161 TAATGAGATT CCTGTCGCTT CCATCCCTGG CCGGCCAGCG 201 GGCGGGCTCC CCAGCCAGGC CGCTGCACCT GTCAGGGGAA 241 CAAGCTGGAG GAGCAGGACC CTAGACCTCT GCAGCCCATA 281 CCAGGTCTCA TGGAGGGGAA CAAGCTGGAG GAGCAGGACT 321 CTAGCCCTCC ACAGTCCACT CCAGGGCTCA TGAAGGGGAA 361 CAAGCGTGAG GAGCAGGGGC TGGGCCCCGA ACCTGCGGCG 401 CCCCAGCAGC CCACGGCGGA GGAGGAGGCC CTGATCGAGT 441 TCCACCGCTC CTACCGAGAG CTCTTCGAGT TCTTCTGCAA 481 CAACACCACC ATCCACGGCG CCATCCGCCT GGTGTGCTCC 521 CAGCACAACC GCATGAAGAC GGCCTTCTGG GCAGTGCTGT 561 GGCTCTGCAC CTTTGGCATG ATGTACTGGC AATTCGGCCT 601 GCTTTTCGGA GAGTACTTCA GCTACCCCGT CAGCCTCAAC 641 ATCAACCTCA ACTCGGACAA GCTCGTCTTC CCCGCAGTGA 681 CCATCTGCAC CCTCAATCCC TACAGGTACC CGGAAATTAA 721 AGAGGAGCTG GAGGAGCTGG ACCGCATCAC AGAGCAGACG 761 CTCTTTGACC TGTACAAATA CAGCTCCTTC ACCACTCTCG 801 TGGCCGGCTC CCGCAGCCGT CGCGACCTGC GGGGGACTCT 841 GCCGCACCCC TTGCAGCGCC TGAGGGTCCC GCCCCCGCCT 881 CACGGGGCCC GTCGAGCCCG TAGCGTGGCC TCCAGCTTGC 921 GGGACAACAA CCCCCAGGTG GACTGGAAGG ACTGGAAGAT 961 CGGCTTCCAG CTGTGCAACC AGAACAAATC GGACTGCTTC 1001 TACCAGACAT ACTCATCAGG GGTGGATGCG GTGAGGGAGT 1041 GGTACCGCTT CCACTACATC AACATCCTGT CGAGGCTGCC 1081 AGAGACTCTG CCATCCCTGG AGGAGGACAC GCTGGGCAAC 1121 TTCATCTTCG CCTGCCGCTT CAACCAGGTC TCCTGCAACC 1161 AGGCGAATTA CTCTCACTTC CACCACCCGA TGTATGGAAA 1201 CTGCTATACT TTCAATGACA AGAACAACTC CAACCTCTGG 1241 ATGTCTTCCA TGCCTGGAAT CAACAACGGT CTGTCCCTGA 1281 TGCTGCGCGC AGAGCAGAAT GACTTCATTC CCCTGCTGTC 1321 CACAGTGACT GGGGCCCGGG TAATGGTGCA CGGGCAGGAT 1361 GAACCTGCCT TTATGGATGA TGGTGGCTTT AACTTGCGGC 1401 CTGGCGTGGA GACCTCCATC AGCATGAGGA AGGAAACCCT 1441 GGACAGACTT GGGGGCGATT ATGGCGACTG CACCAAGAAT 1481 GGCAGTGATG TTCCTGTTGA GAACCTTTAC CCTTCAAAGT 1521 ACACACAGCA GGTGTGTATT CACTCCTGCT TCCAGGAGAG 1561 CATGATCAAG GAGTGTGGCT GTGCCTACAT CTTCTATCCG 1601 CGGCCCCAGA ACGTGGAGTA CTGTGACTAC AGAAAGCACA 1641 GTTCCTGGGG GTACTGCTAC TATAAGCTCC AGGTTGACTT 1681 CTCCTCAGAC CACCTGGGCT GTTTCACCAA GTGCCGGAAG 1721 CCATGCAGCG TGACCAGCTA CCAGCTCTCT GCTGGTTACT 1761 CACGATGGCC CTCGGTGACA TCCCAGGAAT GGGTCTTCCA 1801 GATGCTATCG CGACAGAACA ATTACACCGT CAACAACAAG 1841 AGAAATGGAG TGGCCAAAGT CAACATCTTC TTCAAGGAGC 1881 TGAACTACAA AACCAATTCT GAGTCTCCCT CTGTCACGAT 1921 GGTCACCCTC CTGTCCAACC TGGGCAGCCA GTGGAGCCTG 1961 TGGTTCGGCT CCTCGGTGTT GTCTGTGGTG GAGATGGCTG 2001 AGCTCGTCTT TGACCTGCTG GTCATCATGT TCCTCATGCT 2041 GCTCCGAAGG TTCCGAAGCC GATACTGGTC TCCAGGCCGA 2081 GGGGGCAGGG GTGCTCAGGA GGTAGCCTCC ACCCTGGCAT 2121 CCTCCCCTCC TTCCCACTTC TGCCCCCACC CCATGTCTCT 2161 GTCCTTGTCC CAGCCAGGCC CTGCTCCCTC TCCAGCCTTG 2201 ACAGCCCCTC CCCCTGCCTA TGCCACCCTG GGCCCCCGCC 2241 CATCTCCAGG GGGCTCTGCA GGGGCCAGTT CCTCC A CCTG 2281 TCCTCTGGGG GGGCCCTGAG AGGGAAGGAG AGGTTTCTCA 2321 CACCAAGGCA GATGCTCCTC TGGTGGGAGG GTGCTGGCCC 2361 TGGCAAGATT GAAGGATGTG CAGGGCTTCC TCTCAGAGCC 2401 GCCCAAACTG CCGTTGATGT GTGGAGGGGA AGCAAGATGG 2441 GTAAGGGCTC AGGAAGTTGC TCCAAGAACA GTAGCTGATG 2481 AAGCTGCCCA GAAGTGCCTT GGCTCCAGCC CTGTACCCCT 2521 TGGTACTGCC TCTGAACACT CTGGTTTCCC CACCCAACTG 2561 CGGCTAAGTC TCTTTTTCCC TTGGATCAGC CAAGCGAAAC 2601 TTGGAGCTTT GACAAGGAAC TTTCCTAAGA AACCGCTGAT 2641 AACCAGGACA AAACACAACC AAGGGTACAC GCAGGCATGC 2681 ACGGGTTTCC TGCCCAGCGA CGGCTTAAGC CAGCCCCCGA 2721 CTGGCCTGGC CACACTGCTC TCCAGTAGCA CAGATGTCTG 2761 CTCCTCCTCT TGAACTTGGG TGGGAAACCC CACCCAAAAG 2801 CCCCCTTTGT TACTTAGGCA ATTCCCCTTC CCTGACTCCC 2841 GAGGGCTAGG GCTAGAGCAG ACCCGGGTAA GTAAAGGCAG 2881 ACCCAGGGCT CCTCTAGCCT CATACCCGTG CCCTCACAGA 2921 GCCATGCCCC GGCACCTCTG CCCTGTGTCT TTCATACCTC 2961 TACATGTCTG CTTGAGATAT TTCCTCAGCC TGAAAGTTTC 3001 CCCAACCATC TGCCAGAGAA CTCCTATGCA TCCCTTAGAA 3041 CCCTGCTCAG ACACCATTAC TTTTGTGAAC GCTTCTGCCA 3081 CATCTTGTCT TCCCCAAAAT TGATCACTCC GCCTTCTCCT 3121 GGGCTCCCGT AGCACACTAT AACATCTGCT GGAGTGTTGC 3161 TGTTGCACCA TACTTTCTTG TACATTTGTG TCTCCCTTCC 3201 CAACTAGACT GTAAGTGCCT TGCGGTCAGG GACTGAATCT 3241 TGCCCGTTTA TGTATGCTCC ATGTCTAGCC CATCATCCTG 3281 CTTGGAGCAA GTAGGCAGGA GCTCAATAAA TGTTTGTTGC 3321 ATGAAGGAAA AAAAAAAAAA AAAAA

The human epithelial Na+channel encoded by the SCNN1A cDNA with SEQ ID NO:24 has the following sequence (SEQ ID NO:25).

1 MEGNKLEEQD SSPPQSTPGL MKGNKREEQG LGPEPAAPQQ 41 PTAEEEALIE FHRSYRELFE FFCNNTTIHG AIRLVCSQHN 81 RMKTAFWAVL WLCTFGMMYW QFGLLFGEYF SYPVSLNINL 121 NSDKLVFPAV TICTLNPYRY PEIKEELEEL DRITEQTLFD 161 LYKYSSFTTL VAGSRSRRDL RGTLPHPLQR LRVPPPPHGA 201 RRARSVASSL RDNNPQVDWK DWKIGFQLCN QNKSDCFYQT 241 YSSGVDAVRE WYRFHYINIL SRLPETLPSL EEDTLGNFIF 281 ACRFNQVSCN QANYSHFHHP MYGNCYTFND KNNSNLWMSS 321 MPGINNGLSL MLRAEQNDFI PLLSTVTGAR VMVHGQDEPA 361 FMDDGGFNLR PGVETSISMR KETLDRLGGD YGDCTKNGSD 401 VPVENLYPSK YTQQVCIHSC FQESMIKECG CAYIFYPRPQ 441 NVEYCDYRKH SSWGYCYYKL QVDFSSDHLG CFTKCRKPCS 481 VTSYQLSAGY SRWPSVTSQE WVFQMLSRQN NYTVNNKRNG 521 VAKVNIFFKE LNYKTNSESP SVTMVTLLSN LGSQWSLWFG 561 SSVLSVVEMA ELVFDLLVIM FLMLLRRFRS RYWSPGRGGR 601 GAQEVASTLA SSPPSHFCPH PMSLSLSQPG PAPSPALTAP 641 PPAYATLGPR PSPGGSAGAS SS T CPLGGP

Note that the underlined threonine at position 722 of the SEQ ID NO:23 SCNN1A protein, and the underlined threonine at position 663 of the SEQ ID NO:25 SCNN1A protein, is threonine because some individuals have nucleotide sequence SEQ ID NO:22, where the variable nucleotide is adenine. However, position 722 of SEQ ID NO:23 and position 663 of SEQ ID NO:25 can be alanine in some individuals because those individuals have guanine as the variable nucleotide in sequence SEQ ID NO:22.

Patients with the threonine substitution in SCNN1A (adenine in rs2228576) have more functional Na⁺ channels and consequently higher activity higher voltage currents across the cells. Hence, patients with such a threonine at the variable site in SCNN1A are more susceptible to hypertension than SCNNIA proteins with alanine at that position. Patients with the threonine substitution in SCNN1A can benefit from administration of amiloride.

Common and functional genetic variation of alpha adducin at amino acid 460 has also been identified where some individuals have glycine and others have tryptophan. A cDNA sequence for the human alpha adducin gene (ADD1) is available from the NCBI database as accession number NM_001119.4 (GI:346644753). This ADD1 sequence is provided below as SEQ ID NO:26.

1 GCACCCAGGT CGGGCGGTGG GGGCGAGCGG AGGGGCTGAG 41 GGGCGGAGAG GCCTGGCGGG CCGCTGCTGC GGGCCAGGGG 81 ACGGGGGCGG AGCCGGAGCC GGAGCCGACG GGCGGTGGCC 121 GCACTGGGAC CCCGGAATCC CGCGCGCTGC CCACGATTCG 161 CTTCTGAGGA ACCTAGAAAG ATTGTACAAT GAATGGTGAT 201 TCTCGTGCTG CGGTGGTGAC CTCACCACCC CCGACCACAG 241 CCCCTCACAA GGAGAGGTAC TTCGACCGAG TAGATGAGAA 281 CAACCCAGAG TACTTGAGGG AGAGGAACAT GGCACCAGAC 321 CTTCGCCAGG ACTTCAACAT GATGGAGCAA AAGAAGAGGG 361 TGTCCATGAT TCTGCAAAGC CCTGCTTTCT GTGAAGAATT 401 GGAATCAATG ATACAGGAGC AATTTAAGAA GGGGAAGAAC 441 CCCACAGGCC TATTGGCATT ACAGCAGATT GCAGATTTTA 481 TGACCACGAA TGTACCAAAT GTCTACCCAG CAGCTCCGCA 521 AGGAGGGATG GCTGCCTTAA ACATGAGTCT TGGTATGGTG 561 ACTCCTGTGA ACGATCTTAG AGGATCTGAT TCTATTGCGT 601 ATGACAAAGG AGAGAAGTTA TTACGGTGTA AATTGGCAGC 641 GTTTTATAGA CTAGCAGATC TCTTTGGGTG GTCTCAGCTT 681 ATCTACAATC ATATCACAAC CAGAGTGAAC TCCGAGCAGG 721 AACACTTCCT CATTGTCCCT TTTGGGCTTC TTTACAGTGA 761 AGTGACTGCA TCCAGTTTGG TTAAGATCAA TCTACAAGGA 801 GATATAGTAG ATCGTGGAAG CACTAATCTG GGAGTGAATC 841 AGGCCGGCTT CACCTTACAC TCTGCAATTT ATGCTGCACG 881 CCCGGACGTG AAGTGCGTCG TGCACATTCA CACCCCAGCA 921 GGGGCTGCGG TCTCTGCAAT GAAATGTGGC CTCTTGCCAA 961 TCTCCCCGGA GGCGCTTTCC CTTGGAGAAG TGGCTTATCA 1001 TGACTACCAT GGCATTCTGG TTGATGAAGA GGAAAAAGTT 1041 TTGATTCAGA AAAATCTGGG GCCTAAAAGC AAGGTTCTTA 1081 TTCTCCGGAA CCATGGGCTC GTGTCAGTTG GAGAGAGCGT 1121 TGAGGAGGCC TTCTATTACA TCCATAACCT TGTGGTTGCC 1161 TGTGAGATCC AGGTTCGAAC TCTGGCCAGT GCAGGAGGAC 1201 CAGACAACTT AGTCCTGCTG AATCCTGAGA AGTACAAAGC 1241 CAAGTCCCGT TCCCCAGGGT CTCCGGTAGG GGAAGGCACT 1281 GGATCGCCTC CCAAGTGGCA GATTGGTGAG CAGGAATTTG 1321 AAGCCCTCAT GCGGATGCTC GATAATCTGG GCTACAGAAC 1361 TGGCTACCCT TATCGATACC CTGCTCTGAG AGAGAAGTCT 1401 AAAAAATACA GCGATGTGGA GGTTCCTGCT AGTGTCACAG 1441 GTTACTCCTT TGCTAGTGAC GGTGATTCGG GCACTTGCTC 1481 CCCACTCAGA CACAGTTTTC AGAAGCAGCA GCGGGAGAAG 1521 ACAAGATGGC TGAACTCTGG CCGGGGCGAC GAAGCTTCCG 1561 AGGAA G GGCA GAATGGAAGC AGTCCCAAGT CGAAGACTAA 1601 GTGGACTAAA GAGGATGGAC ATAGAACTTC CACCTCTGCT 1641 GTCCCTAACC TGTTTGTTCC ATTGAACACT AACCCAAAAG 1681 AGGTCCAGGA GATGAGGAAC AAGATCCGAG AGCAGAATTT 1721 ACAGGACATT AAGACGGCTG GCCCTCAGTC CCAGGTTTTG 1761 TGTGGTGTAG TGATGGACAG GAGCCTCGTC CAGGGAGAGC 1801 TGGTGACGGC CTCCAAGGCC ATCATTGAAA AGGAGTACCA 1841 GCCCCACGTC ATTGTGAGCA CCACGGGCCC CAACCCCTTC 1881 ACCACACTCA CAGACCGTGA GCTGGAGGAG TACCGCAGGG 1921 AGGTGGAGAG GAAGCAGAAG GGCTCTGAAG AGAATCTGGA 1961 CGAGGCTAGA GAACAGAAAG AAAAGAGTCC TCCAGACCAG 2001 CCTGCGGTCC CCCACCCGCC TCCCAGCACT CCCATCAAGC 2041 TGGAGGAAGA CCTTGTGCCG GAGCCGACTA CTGGAGATGA 2081 CAGTGATGCT GCCACCTTTA AGCCAACTCT CCCCGATCTG 2121 TCCCCTGATG AACCTTCAGA AGCACTCGGC TTCCCAATGT 2161 TAGAGAAGGA GGAGGAAGCC CATAGACCCC CAAGCCCCAC 2201 TGAGGCCCCT ACTGAGGCCA GCCCCGAGCC AGCCCCAGAC 2241 CCAGCCCCGG TGGCTGAAGA GGCTGCCCCC TCAGCTGTCG 2281 AGGAGGGGGC CGCCGCGGAC CCTGGCAGCG ATGGGTCTCC 2321 AGGCAAGTCC CCGTCCAAAA AGAAGAAGAA GTTCCGTACC 2361 CCGTCCTTTC TGAAGAAGAG CAAGAAGAAG AGTGACTCCT 2401 GAAAGCCCTG CGCTAACACT GTCCTGTCCG GAGCGACCCT 2441 GGCTCTGCCA GCGTCCCCGG CCACGTCTGT GCTCTGTCCT 2481 TGTGTAATGG AATGCAAAAA AGCCAAGCCC TCCGCCTAGA 2521 GGTCCCCTCA CGTGACCAGC CCCGTGTAGC CCCGGGCTGA 2561 CCCAGTGTGT GCTCAGCAGC CCCACCCCAC CCTGCCCCTT 2601 GTCCTCTCAG AGCCTCAGCT TCTGGGGGAG ACATGCTCTC 2641 CCCACAGGGG GGAGGCACTA AGTCATGGTC CTGGCTGGAA 2681 GGTACTGAAG GCTTCTGCAG CTTTGGCTGC ACGTCACCCT 2721 CCTGAGCCTC ACCTTTCCTG CCGTCCCTCC TGTTGTGAAA 2761 TCACCACATT CTGTCTCTGC TTGGCTTCCC CTCCACCCTA 2801 AAGTCTCAGG TGACGGACTC AGACTCCTGG CTTCATGTGG 2841 CATTCTCTCT GCTCAGTGAT CTCACTTAAA TCTATATACA 2881 AAGCCTTGGT CCCGTGAAAA CACTCGTGTG CCCACCAGCG 2921 GCCTTGAAGA GGCAGGTCTG GGCCAGATGC TGGGCAGGAA 2961 ACCCCAGCGG CAGATGGGCC TGTGTGCACC CAACGTGATG 3001 CTATGCATGT CTGACCGACG ATCCCTCGAC CAGAATCAGA 3041 TTCAGGAGCT CAGTTTCTTT TTCACTTGGG TCTCTGGATT 3081 CCTGTCATAG GGAAGGTATA TCAGGAGGGG AAGAGGCCTT 3121 TCTAGAATTT TCTTTGAGCA GGTTTACAAT TTAGCTTACA 3161 TTTTTCGACT GTGAACGTGA ATAGGCTGCT TTTTGCTTTC 3201 TTCTTTCCAG ACCCCACAGT AGAGCACTTT TCACTTATTT 3241 GGGGGAGGCT TCAGGGGACT GTTCTCACCT TAACTCAGCC 3281 AGAAAGATGC CCTAGTTGTG ATCAAAGGTA ACTCGAGGTG 3321 GAGGGTAGCC CTGGGGCCCC TCGACATCAC CGTCATTGAT 3361 GGAGCCTGAA CCGTGTGCTC CTCGGCAGAT GCTGTTGTTG 3401 TTACTTCCCT CCAAGAGGCT GGAAAAGGGC TCAGAGCTGC 3441 TGAGCAGGAA CCGGAGGGTG ACCCATTTCA GGAGGTGCCG 3481 GTACCAGCCT GACTAGGTAC AGGCAAGCTT GTGTGGGCCC 3521 AACAGGCCCT TGGTAGAGCT GGTGCCAGAT GTGGGCTCAG 3561 ATCCTGGGCA TGATGGGCCG AGCCACCTCG GATCCCACTG 3601 ATTGGCCAGC CGAGCGAGAA CCAGGCTGCT GCATGGCACT 3641 GACCGCCGCT TCCAGCTTCC TCTGAGCCGC AGGGCCTGCT 3681 ACGCGGGCAA GCGTGCTGCC TCTCTTCTGT GTCGTTTTGT 3721 TGCCAAGGCA GAATGAAAAG TCCTTAACCG TGGACTCTTC 3761 CTTTATCCCC TCCTTTACCC CACATATGCA ATGACTTTTA 3801 ATTTTCACTT TTGTAGTTTA ATCCTTTGTA TTACAACATG 3841 AAATATAGTT GCATATATGG ACACCGACTT GGGAGGACAG 3881 GTCCTGAATG TCCTTTCTCC AGTGTAACAT GTTTTACTCA 3921 CAAATAAAAT TCTTTCAGCA AGTTCCTTGT CTAAAAAAAA 3961 AAAAAAAAAA

The rs4961 single nucleotide polymorphism (SNP) is present in the ADD1 gene, where the variable nucleotide is at about position 1566 in SEQ ID NO:26 (underlined), which can be guanine in some individuals and thymine in others. The rs4961 sequence (SEQ ID NO:27) is shown below, where the underlined G/T is the SNP.

CCGGGGCGACGAAGCTTCCGAGGAA [G/T] GGCAGAATGGAAGCAGTCCC AAGTC.

The human alpha adducin encoded by the ADD1 cDNA with SEQ ID NO:26 has the following sequence (SEQ ID NO:28).

1 MNGDSRAAVV TSPPPTTAPH KERYFDRVDE NNPEYLRERN 41 MAPDLRQDFN MMEQKKRVSM ILQSPAFCEE LESMIQEQFK 81 KGKNPTGLLA LQQIADFMTT NVPNVYPAAP QGGMAALNMS 121 LGMVTPVNDL RGSDSIAYDK GEKLLRCKLA AFYRLADLFG 161 WSQLIYNHIT TRVNSEQEHF LIVPFGLLYS EVTASSLVKI 201 NLQGDIVDRG STNLGVNQAG FTLHSAIYAA RPDVKCVVHI 241 HTPAGAAVSA MKCGLLPISP EALSLGEVAY HDYHGILVDE 281 EEKVLIQKNL GPKSKVLILR NHGLVSVGES VEEAFYYIHN 321 LVVACEIQVR TLASAGGPDN LVLLNPEKYK AKSRSPGSPV 361 GEGTGSPPKW QIGEQEFEAL MRMLDNLGYR TGYPYRYPAL 401 REKSKKYSDV EVPASVTGYS FASDGDSGTC SPLRHSFQKQ 441 QREKTRWLNS GRGDEASEE G  QNGSSPKSKT KWTKEDGHRT 481 STSAVPNLFV PLNTNPKEVQ EMRNKIREQN LQDIKTAGPQ 521 SQVLCGVVMD RSLVQGELVT ASKAIIEKEY QPHVIVSTTG 561 PNPFTTLTDR ELEEYRREVE RKQKGSEENL DEAREQKEKS 601 PPDQPAVPHP PPSTPIKLEE DLVPEPTTGD DSDAATFKPT 641 LPDLSPDEPS EALGFPMLEK EEEAHRPPSP TEAPTEASPE 681 PAPDPAPVAE EAAPSAVEEG AAADPGSDGS PGKSPSKKKK 721 KFRTPSFLKK SKKKSDS

Note that the underlined glycine at position 460 of the SEQ ID NO:28 alpha adducin protein is glycine because some individuals have nucleotide sequence SEQ ID NO:26, where the variable nucleotide at position 1566 is guanine. However, position 460 of SEQ ID NO:28 can be tryptophan in some individuals because those individuals have thymine as the variable nucleotide at position 1566 in sequence SEQ ID NO:28.

Individuals with the tryptophan variant of alpha adducin are more likely to be salt sensitive, more likely to have hypertension and have a greater response to diuretics.

Genetic variation of the sodium (Na⁺) chloride (Cl⁻) co-transporter (SLC12A3) also has blood pressure consequences. A cDNA sequence for the sodium (Na⁺) chloride (Cl⁻) co-transporter (SLC12A3) is available from the NCBI database as accession number NM_000339.2 (GI: 186910314). This SLC12A3 cDNA sequence is provided below as SEQ ID NO:29.

1 CTGGCCCCTC CCTGGACACC CAGGCGACAA TGGCAGAACT 41 GCCCACAACA GAGACGCCTG GGGACGCCAC TTTGTGCAGC 81 GGGCGCTTCA CCATCAGCAC ACTGCTGAGC AGTGATGAGC 121 CCTCTCCACC AGCTGCCTAT GACAGCAGCC ACCCCAGCCA 161 CCTGACCCAC AGCAGCACCT TCTGCATGCG CACCTTTGGC 201 TACAACACGA TCGATGTGGT GCCCACATAT GAGCACTATG 241 CCAACAGCAC CCAGCCTGGT GAGCCCCGGA AGGTCCGGCC 281 CACACTGGCT GACCTGCACT CCTTCCTCAA GCAGGAAGGC 321 AGACACCTGC ATGCCCTGGC CTTTGACAGC CGGCCCAGCC 361 ACGAGATGAC TGATGGGCTG GTGGAGGGCG AGGCAGGCAC 401 CAGCAGCGAG AAGAACCCCG AGGAGCCAGT GCGCTTCGGC 441 TGGGTCAAGG GGGTGATGAT TCGTTGCATG CTCAACATTT 481 GGGGCGTGAT CCTCTACCTG CGGCTGCCCT GGATTACGGC 521 CCAGGCAGGC ATCGTCCTGA CCTGGATCAT CATCCTGCTG 561 TCGGTCACGG TGACCTCCAT CACAGGCCTC TCCATCTCAG 601 CCATCTCCAC CAATGGCAAG GTCAAGTCAG GTGGCACCTA 641 CTTCCTCATC TCCCGGAGTC TGGGCCCAGA GCTTGGGGGC 681 TCCATCGGCC TCATTTTCGC TTTCGCCAAT GCCGTGGGTG 721 TGGCCATGCA CACGGTGGGC TTTGCAGAGA CCGTGCGGGA 761 CCTGCTCCAG GAGTATGGGG CACCCATCGT GGACCCCATT 801 AACGACATCC GCATCATTG G  CGTGGTCTCG GTCACTGTGC 841 TGCTGGCCAT CTCCCTGGCT GGCATGGAGT GGGAGTCCAA 881 GGCCCAGGTG CTGTTCTTCC TTGTCATCAT GGTCTCCTTT 921 GCCAACTATT TAGTGGGGAC GCTGATCCCC CCATCTGAGG 961 ACAAGGCCTC CAAAGGCTTC TTCAGCTACC GGGCGGACAT 1001 TTTTGTCCAG AACTTGGTGC CTGACTGGCG GGGTCCAGAT 1041 GGCACCTTCT TCGGAATGTT CTCCATCTTC TTCCCCTCGG 1081 CCACAGGCAT CCTGGCAGGG GCCAACATAT CTGGTGACCT 1121 CAAGGACCCT GCTATAGCCA TCCCCAAGGG GACCCTCATG 1161 GCCATTTTCT GGACGACCAT TTCCTACCTG GCCATCTCAG 1201 CCACCATTGG CTCCTGCGTG GTGCGTGATG CCTCTGGGGT 1241 CCTGAATGAC ACAGTGACCC CTGGCTGGGG TGCCTGCGAG 1281 GGGCTGGCCT GCAGCTATGG CTGGAACTTC ACCGAGTGCA 1321 CCCAGCAGCA CAGCTGCCAC TACGGCCTCA TCAACTATTA 1361 CCAGACCATG AGCATGGTGT CAGGCTTCGC GCCCCTGATC 1401 ACGGCTGGCA TCTTCGGGGC CACCCTCTCC TCTGCCCTGG 1441 CCTGCCTTGT CTCTGCTGCC AAAGTCTTCC AGTGCCTTTG 1481 CGAGGACCAG CTGTACCCAC TGATCGGCTT CTTCGGCAAA 1521 GGCTATGGCA AGAACAAGGA GCCCGTGCGT GGCTACCTGC 1561 TGGCCTACGC CATCGCTGTG GCCTTCATCA TCATCGCTGA 1601 GCTCAACACC ATAGCCCCCA TCATTTCCAA CTTCTTCCTC 1641 TGCTCCTATG CCCTCATCAA CTTCAGCTGC TTCCACGCCT 1681 CCATCACCAA CTCGCCTGGG TGGAGACCTT CATTCCAATA 1721 CTACAACAAG TGGGCGGCGC TGTTTGGGGC TATCATCTCC 1761 GTGGTCATCA TGTTCCTCCT CACCTGGTGG GCGGCCCTCA 1801 TCGCCATTGG CGTGGTGCTC TTCCTCCTGC TCTATGTCAT 1841 CTACAAGAAG CCAGAGGTAA ATTGGGGCTC CTCGGTACAG 1881 GCTGGCTCCT ACAACCTGGC CCTCAGCTAC TCGGTGGGCC 1921 TCAATGAGGT GGAAGACCAC ATCAAGAACT ACCGCCCCCA 1961 GTGCCTGGTG CTCACGGGGC CCCCCAACTT CCGCCCGGCC 2001 CTGGTGGACT TTGTGGGCAC CTTCACCCGG AACCTCAGCC 2041 TGATGATCTG TGGCCACGTG CTCATCGGAC CCCACAAGCA 2081 GAGGATGCCT GAGCTCCAGC TCATCGCCAA CGGGCACACC 2121 AAGTGGCTGA ACAAGAGGAA GATCAAGGCC TTCTACTCGG 2161 ATGTCATTGC CGAGGACCTC CGCAGAGGCG TCCAGATCCT 2201 CATGCAGGCC GCAGGTCTCG GGAGAATGAA GCCCAACATT 2241 CTGGTGGTTG GGTTCAAGAA GAACTGGCAG TCGGCTCACC 2281 CGGCCACAGT GGAAGACTAC ATTGGCATCC TCCATGATGC 2321 CTTTGATTTC AACTATGGCG TGTGTGTCAT GAGGATGCGG 2361 GAGGGACTCA ACGTGTCCAA GATGATGCAG GCGCACATTA 2401 ACCCCGTGTT TGACCCAGCG GAGGACGGGA AGGAAGCCAG 2441 CGCCAGAGGT GCCAGGCCAT CAGTCTCTGG CGCTTTGGAC 2481 CCCAAGGCCC TGGTGAAGGA GGAGCAGGCC ACCACCATCT 2521 TCCAGTCGGA GCAGGGCAAG AAGACCATAG ACATCTACTG 2561 GCTCTTTGAC GATGGAGGCC TCACCCTCCT CATTCCCTAT 2601 CTCCTTGGCC GCAAGAGGAG GTGGAGCAAA TGCAAGATCC 2641 GTGTGTTCGT AGGCGGCCAG ATTAACAGGA TGGACCAGGA 2681 GAGAAAGGCG ATCATTTCTC TGCTGAGCAA GTTCCGACTG 2721 GGATTCCATG AAGTCCACAT CCTCCCTGAC ATCAACCAGA 2761 ACCCTCGGGC TGAGCACACC AAGAGGTTTG AGGACATGAT 2801 TGCACCCTTC CGTCTGAATG ATGGCTTCAA GGATGAGGCC 2841 ACTGTCAACG AGATGCGGCG GGACTGCCCC TGGAAGATCT 2881 CAGATGAGGA GATTACGAAG AACAGAGTCA AGTCCCTTCG 2921 GCAGGTGAGG CTGAATGAGA TTGTGCTGGA TTACTCCCGA 2961 GACGCTGCTC TCATCGTCAT CACTTTGCCC ATAGGGAGGA 3001 AGGGGAAGTG CCCCAGCTCG CTGTACATGG CCTGGCTGGA 3041 GACCCTGTCC CAGGACCTCA GACCTCCAGT CATCCTGATC 3081 CGAGGAAACC AGGAAAACGT GCTCACCTTT TACTGCCAGT 3121 AACTCCAGGC TTTGACATCC CTGTCCACAG CTCTGAGTGT 3161 GTGGGATAAG TTGGAACTTG ATTGCCTCTA GTCCACAGGG 3201 ATGAGACTCA TGTTCTGTTG CACTTTAAGT GGCAGCATCT 3241 GATGATCTCA CCGAAAAAGA TGGTAGATTT CCAAATCTGG 3281 CTGGACTCCA CTTCCATGGG ACACATTCCC TGGGTCTTGT 3321 GTTTATAGGC TAGAGAAATA GCAGATGGAG CTGCAAGGAA 3361 AACTCTCTAA AGCATCCTAT TCCTTTTAAA GGATTTCTTT 3401 TGATTTTGAT GACCATTAAT TAAGAGTTCA GTCTTTGATT 3441 TGTATGCAAA TTGGAGTCCC AATGCTGGGC GTGAATCTTG 3481 ACAGTTTCTA CAGACCTTCC TGGGTGAAAG TTCCTAAATC 3521 ATGCCCTGCT TCCTCCAATA GGAGAATGGG AGCCTCACCT 3561 GTAGGACCTA CAGGCTCTCT AAGGAATGCA GGTCTCTCTC 3601 TGAGCCTCCA CAGCCAGGCA AATACATATA TATATATTTT 3641 TTTTTTAGAT GAAGTTTTTT CTCTTGTTGC CCAGGCTAGG 3681 GTGTAATGGC ATGATCTCAG GTCACTGCAA CCTCCTCCCG 3721 GGTTCAAGCA TTTCTTCTGT CTCAGCCTCC CGAATAGCTG 3761 GGATTACAGG CACCTGCCAT CACACGAGCT AATTTTTGTA 3801 TTTTTAGTAG AGATGGGGTT TCACCATGTT GACCAGGCTG 3841 GTGTTGAGCT CCTGACCTCA GGTGATCCAC CCACCTCGGT 3881 CTCCCAAAGT GCTGGGGTTA CAGGCCTGAG CCACTGCGCC 3921 CGGCCCAGGC AAATTTCTTG AACCACTTCT CACTCCCGTC 3961 ACTTTCAATA AGGGGTCTTT GATGTCTTCA CTGGTTCTTT 4001 GGACGAGGGA CTTTTCGAAC TTTTTTGGTT GCAACACACA 4041 GTAAGAAATA TACTTCACAC TGAGACTTGC AGCGCACACA 4081 CACGGAAACG ACCAAAACAA AAATGTCACA AAACAATACT 4121 TACCCTTCCC TGGGGGACGT CCTCCAGTAT GTTCTGTTCT 4161 GTTTATTTTT CACTGTTGGT TGCAATCCAA TAAAATGACT 4201 TTGGGATCCA CTCATGGGTG GGGACCCACA CATTTGAAAG 4241 GCATGGCCAC CTTTCTGTTG TGCCTTGCAT TTGTCCACAC 4281 ACAGGGAGTC TGGCTGAGCT GGGGAAAGGC CACGGCTGGG 4321 TGTCATTGCC ATTTTCCCAG CTCATCTCAC CGGGAAGAAA 4361 AGCAGATTGA CAGAACACGT GAGGAGGGGT ATTGATGGCA 4001 GGAGAGTCAA AAAAGAGTTT TAAAGAAGGG GCAAGGTTGA 4441 AGGAGTCTAG TGGCAAGGGT AAGATTTCAG GCATGGTTAA 4481 GAACAGACGA CAAGGATGTC AGGAATGAAG ATGTGGAGAG 4521 GGGTGTAGAG ATGGCAAGGT TGGCAAGGAA CAGATAGGCA 4561 GGAGCAGGTC CAAGCCAAGC CTAGCCCAAG ACCAGGTGAA 4601 AGGAGAGGGG AGGAGGAGCC ACCTGCAAGA GATGGAAAGA 4641 GCAGGCGGCA GAGGGGGCTG GCAGGGAGGG GCTGTTAAGA 4681 GTGGGGTTGG AGGTGGGAGA GAAGCTAGGA CAAGGGAGAT 4721 GGAGAAAGGA CCTATACCTG GCTCACGGAA GGCCTTCAGG 4761 TCACTACACG TTGAACATCC CCAGTGTTTG AGCCCCCAAA 4801 GCTAGGGTGC AAGAGCACTG CCATCGAATG CCAGTGGGTG 4841 AGGCCAAGTG AGGGTATTTG CAGCTCTAGA CATAACCAAG 4881 AAGCGTAAAG GTGAGTTGTT TGGTGGTACG ACTGCCTGTG 4921 CCTTCTTCCG ATGGCACTGG GGTGGCTGAA GGAACAGACA 4961 TCTTTGGGTT TCATCAGCCT CCTCCAAGAC TGCTGCAGTG 5001 CCTACACTTT AGACTTCAGA AGGAGACTAA AGACTTCTAG 5041 AATTTAGAAG GAGATCTGAA GTCTCCTTTC TGGAGTTACA 5081 ACCCAAAGGA TGTTAGCATT TCTCAGGTCA TCCCACTGCA 5121 AAGCCCAGAA GGCTTGGGGC TCCCAGGCTG CTCTGAAGCC 5161 CCACTGTCTG ACCGCCTCAG GGCTTGCTAC GAGGGACTGG 5201 GGCACGGCCA AGCTGACTAG GAACAGCTCT CGTGCTCCTG 5241 AGGGACCTGG AGGATGGGCC TGCCTCCCAG CCATTGAGCT 5281 GGATTCTGGG ATAATTCTTA ACTCGAAATA AGGGGAAGCA 5321 TCCATCAGGG AATGCTGGCC TTTCTAGAGC CACGTAGAAA 5361 ACAATTTTCT GGTTCTTCAA ACCTCAAAGA GTCCTTGGTC 5401 CAAAAAACAG AATGTTTTGG CTTCGGGTGT CAAAAAAAAA 5441 ATTTTCACGA TGTCAGAAAT AGTATGTTTT TAACAATAGT 5481 AATAGCTTTG TAAAAAAATA AAAAGCTTTA ACAGCGAGGC 5521 CATAAACAAT GAAATGAATA AAAACGGTGG TCATTCAGTC 5561 AACGGAAAAA AAAAAAAAAA AA

The rs1529927 single nucleotide polymorphism (SNP) is present in the SLC12A3 gene, where the variable nucleotide is at about position 820 in SEQ ID NO:29 (underlined), which can be guanine in some individuals and cytosine in others. The rs1529927 sequence (SEQ ID NO:30) is shown below, where the underlined C/G is the SNP.

CCCATTAACGACATCCGCATCATTG [C/G] CGTGGTCTCGGTCACTGTG CTGCTG.

The human the sodium (Na⁺) chloride (Cl⁻) co-transporter encoded by the SLC12A3 cDNA with SEQ ID NO:29 has the following sequence (SEQ ID NO:31).

1 MAELPTTETP GDATLCSGRF TISTLLSSDE PSPPAAYDSS 41 HPSHLTHSST FCMRTFGYNT IDVVPTYEHY ANSTQPGEPR 81 KVRPTLADLH SFLKQEGRHL HALAFDSRPS HEMTDGLVEG 121 EAGTSSEKNP EEPVRFGWVK GVMIRCMLNI WGVILYLRLP 161 WITAQAGIVL TWIIILLSVT VTSITGLSIS AISTNGKVKS 201 GGTYFLISRS LGPELGGSIG LIFAFANAVG VAMHTVGFAE 241 TVRDLLQEYG APIVDPINDI RII G VVSVTV LLAISLAGME 281 WESKAQVLFF LVIMVSFANY LVGTLIPPSE DKASKGFFSY 321 RADIFVQNLV PDWRGPDGTF FGMFSIFFPS ATGILAGANI 361 SGDLKDPAIA IPKGTLMAIF WTTISYLAIS ATIGSCVVRD 401 ASGVLNDTVT PGWGACEGLA CSYGWNFTEC TQQHSCHYGL 441 INYYQTMSMV SGFAPLITAG IFGATLSSAL ACLVSAAKVF 481 QCLCEDQLYP LIGFFGKGYG KNKEPVRGYL LAYAIAVAFI 521 IIAELNTIAP IISNFFLCSY ALINFSCFHA SITNSPGWRP 561 SFQYYNKWAA LFGAIISVVI MFLLTWWAAL IAIGVVLFLL 601 LYVIYKKPEV NWGSSVQAGS YNLALSYSVG LNEVEDHIKN 641 YRPQCLVLTG PPNFRPALVD FVGTFTRNLS LMICGHVLIG 681 PHKQRMPELQ LIANGHTKWL NKRKIKAFYS DVIAEDLRRG 721 VQILMQAAGL GRMKPNILVV GFKKNWQSAH PATVEDYIGI 761 LHDAFDFNYG VCVMRMREGL NVSKMMQAHI NPVFDPAEDG 801 KEASARGARP SVSGALDPKA LVKEEQATTI FQSEQGKKTI 841 DIYWLFDDGG LTLLIPYLLG RKRRWSKCKI RVFVGGQINR 881 MDQERKAIIS LLSKFRLGFH EVHILPDINQ NPRAEHTKRF 921 EDMIAPFRLN DGFKDEATVN EMRRDCPWKI SDEEITKNRV 961 KSLRQVRLNE IVLDYSRDAA LIVITLPIGR KGKCPSSLYM 1001 AWLETLSQDL RPPVILIRGN QENVLTFYCQ

Note that the underlined glycine at position 264 of the SEQ ID NO:31 sodium (Na⁺) chloride (Cl⁻) co-transporter protein is glycine because some individuals have nucleotide sequence SEQ ID NO:29, where the variable nucleotide at position 820 is guanine. However, position 264 of SEQ ID NO:29 can be alanine in some individuals because those individuals have cytosine as the variable nucleotide at position 820 in sequence SEQ ID NO:29.

Patients with the alanine variant of SLC12A3 (encoded by the rs1529927 site (SEQ ID NO:30)) exhibit a stronger diuretic effect to loop diuretics and demonstrate more excretion of Cl⁻ and K⁺ in response to therapy. Hence, subject with alanine or guanine at the rs1529927 site are more response to diuretics.

The WNK1 gene has functional and common polymorphisms that affect how a subject's blood pressure responds to drugs. Several cDNA sequences for the WNT1 gene are available from the NCBI database.

The rs2107614 single nucleotide polymorphism (SNP) is present in an intron of the WNK1 gene, where the variable nucleotide can be thymine in some individuals and cytosine in others. The rs2107614 sequence (SEQ ID NO:33) is shown below, where the underlined C/T is the SNP.

CACTTCCTCCAAAAAAAAAGAAAAC [C/T] CCATTTCCCCTCAACTCTT CCAGTT.

Another SNP, rs1159744, is present an intron of the WNK1 gene, where the variable nucleotide can be guanine in some individuals and cytosine in others. The rs1159744 sequence (SEQ ID NO:34) is shown below, where the underlined C/G is the SNP.

AATGTTAACAGTATAGAAAATTTTA [C/G] CTCAACAAATAGAGAATAT CAGTAA.

Patients with the cytosine variant of WNK1 at SNP positions rs1159744 and rs2107614 exhibit greater blood pressure reductions in response to loop diuretic therapy when compared to patients with the guanine or thymine variants at these two sites, respectively (Turner et al., Hypertension 46:758-765 (2005)).

Therapy

The methods, reagents, devices, and kits described herein can be used for determining whether a subject may benefit from treatment with a blood pressure medication, and which medication can be more effective for treating high blood pressure. For example, the methods described herein can be employed for determining whether a subject should be treated with a diuretic, an angiotensin converting enzyme (ACE) inhibitor, or a beta-blocker. Such determination is performed by identifying or detecting whether the subject has a genetic variant or single nucleotide polymorphism in his or her ADRB1, ADRB2, CYP2D6, angiotensin converting enzyme (ACE), angiotensinogen, angiotensin receptors, renin, Na⁺ channels (such as SCNN1A), adducin, sodium (Na⁺) chloride (Cl⁻) co-transporters (such as SLC12A3), and/or WNK1 polypeptides or nucleic acids. If testing of the subject's tissue sample shows that the subject has a genetic variant or single nucleotide polymorphism in his or her ADRB1, ADRB2, CYP2D6, angiotensin converting enzyme (ACE), angiotensinogen, angiotensin receptors, renin, Na⁺ channels (such as SCNN1A), adducin, sodium (Na⁺) chloride (Cl⁻) co-transporters (such as SLC12A3), and/or WNK1 polypeptides or nucleic acids, a suitable therapeutic regimen can be prescribed for the subject.

A diuretic promotes the production or urine. Diuretics are sometimes grouped into three categories: thiazides, loop, and potassium-sparing diuretics. Thiazide diuretics include chlorothiazide, hydrochlorothiazide, indapamide, metolazone, and chlorthalidone. Loop diuretics include furosemide, bumetanide, ethacrynic acid, and torsemide. Examples of potassium-sparing diuretics include amiloride, eplerenone, spironolactone, and triamterene.

Examples of diuretics that can be employed also include furosemide, thiazides, carbonic anhydrase inhibitors, potassium-sparing diuretics (e.g., aldosterone antagonists, spironolactone, eplerenone, potassium canreonate, amiloride, triamterene, aldactone, and combinations thereof), calcium-sparing diuretics, For example, the diuretic can be acetazolamide, amiloride, bumetanide, chlorothalidone, chlorothiazide, ethacrynic acid, furosemide, glycerin, hydrochlorothiazide, hydroflumthiazide, indapamide, isosorbide, mannitol, methazolamide, methylchlothiazide, metolazone, dichlorphenamide, spironolactone, torsemide, triamterene, urea, and combinations thereof.

The angiotensin converting enzyme inhibitor can be selected from enalapril, lisinopril, captopril alacipril, benazapril, cilazapril, delapril, fosinopril, perindopril, quinapril, ramipril, moveltipril, spirapril, ceronapril, imidapril, temocapril, trandolopril, utilbapril, zofenopril, CV5975, EMD 56855, libenzapril, zalicipril, HOE065, MDL 27088, AB47, DU 1777, MDL 27467A, Equaten™, Prentyl™, Synecor™, and Y23785; and the diuretic is selected from hydrochlorothiazide (HCTZ), furosemide, altizide, trichlormethazide, triflumethazide, bemetizide, cyclothiazide, methylchlothiazide, azosemide, chlorothiazide, butizide, bendroflumethazide, cyclopenthiazide, benzclortriazide, polythiazide, hydroflumethazide, benzthiazide, ethiazide, penflutazide, and any combination thereof.

The angiotensin II receptor antagonists can, for example, be losartan, valsartan, candesartan, irbesartan, olmesartan, or any combination thereof.

The renin inhibitors can be urea derivatives of di- and tri-peptides (See U.S. Pat. No. 5,116,835), amino acids and derivatives (U.S. Pat. Nos. 5,095,119 and 5,104,869), amino acid chains linked by non-peptidic bonds (U.S. Pat. No. 5,114,937), di- and tri-peptide derivatives (U.S. Pat. No. 5,106,835), peptidyl amino diols (U.S. Pat. Nos. 5,063,208 and 4,845,079) and peptidyl beta-aminoacyl aminodiol carbamates (U.S. Pat. No. 5,089,471); also, a variety of other peptide analogs as disclosed in the following U.S. Pat. Nos. 5,071,837; 5,064,965; 5,063,207; 5,036,054; 5,036,053; 5,034,512 and 4,894,437, and small molecule renin inhibitors (including diol sulfonamides and sulfinyls (U.S. Pat. No. 5,098,924), N-morpholino derivatives (U.S. Pat. No. 5,055,466), N-heterocyclic alcohols (U.S. Pat. No. 4,885,292) and pyrolimidazolones (U.S. Pat. No. 5,075,451); also, pepstatin derivatives (U.S. Pat. No. 4,980,283) and fluoro- and chloro-derivatives of statone-containing peptides (U.S. Pat. No. 5,066,643), enalkrein, RO 42-5892, A 65317, CP 80794, ES1005, ES 8891, SQ 34017, aliskiren ((2S,4S,5S,7S)—N-(2-carbamoyl-2-methylpropyl)-5-amino-4-hydroxy-2,7-diisopropyl-8-[4-methoxy-3-(3-methoxypropoxy)phenyl]-oetanamid hemifumarate) SPP600, SPP630 and SPP635), or any combination thereof.

Other therapeutic agents can also be administered including endothelin receptors antagonists, vasodilators, calcium channel blockers (e.g., amlodipine, nifedipine, veraparmil, diltiazem, gallopamil, nifedipine, nimodipins, nicardipine), potassium channel activators (e.g., nicorandil, pinacidil, cromakalim, minoxidil, aprilkalim, loprazolam), diuretics (e.g., hydrochlorothiazide), sympatholitics, beta-adrenergic blocking drugs (e.g., propranolol, atenolol, bisoprolol, carvedilol, metoprolol, or metoprolol tartrate), alpha adrenergic blocking drugs (e.g., doxazocin, prazocin or alpha methyldopa) central alpha adrenergic agonists, peripheral vasodilators (e.g. hydralazine), lipid lowering agents (e.g., simvastatin, lovastatin, ezetamibe, atorvastatin, pravastatin), metabolic altering agents including insulin sensitizing agents and related compounds (e.g., muraglitazar, glipizide, metformin, rosiglitazone) or with other drugs beneficial for the prevention or the treatment of disease including nitroprusside and diazoxide.

The therapeutic protocol can generally be conducted as follows. An assay of all sixteen genotypes (polymorphic sites) can be performed. The therapeutic decision tree of the results can be as follows.

To ascertain whether a diuretic should be administered to a subject, the following can be performed.

-   -   If the subject is (a) homozygous for cytosine at the rs1529927         (SEQ ID NO:30) variable site (expressing alanine at position 264         of the SLC12A3 gene product); (b) homozygous for adenine at the         rs2228576 (SEQ ID NO:22) variable site (expressing threonine at         about 663 or 722 of the SCNN1A protein); and/or (c) homozygous         for thymine at the rs4961 (SEQ ID NO:27) variable site         (expressing tryptophan at about position 460 of the adducin         protein) then the patient should initially start with a diuretic         as the first line of therapy. If the patient is heterozygous at         these sites, then genetic variation within the polymorphic sites         relating to vasodilator and beta-blocker drug class responses         should initially be considered.     -   If the patient does not carry homozygous variants that are known         to be functionally important within the vasodilator and         beta-blocker classes, but are heterozygous at rs1529927,         rs2228576, and rs4961, then diuretic therapy should initially be         considered as first-line therapy.     -   If the subject is homozygous for cytosine at the WNK1 rs1159744         (SEQ ID NO:34) variable site and also homozygous for cytosine at         the WNK1 rs2107614 (SEQ ID NO:33) variable site then the patient         should start with a loop diuretic as first-line of therapy.     -   If the patient does not carry homozygous variants within the         vasodilator and beta-blocker classes that are known to be         functionally important, but are heterozygous at rs1529927,         rs2228576, and rs4961, then loop diuretic therapy should         initially be considered as first-line therapeutic agent.

To ascertain whether a vasodilator should be administered to a subject the following can be performed.

-   -   If the subject is homozygous for cytosine at the rs5186 (SEQ ID         NO:16) variable site of AGT1R, and the subject is homozygous         cytosine at the rs12750834 (SEQ ID NO:20) variable site of         renin, then the patient should use an angiotensin II (AII)         receptor blocker as a first line of therapy.     -   If the patient is heterozygous for cytosine at the rs5186 and         rs12750834 variable sites, but does not present with other         important functional genotypes within the diuretic and         beta-blockade classes, then the patient should also use an         angiotensin II receptor blocker as a first line of therapy.     -   If the patient is homozygous for cytosine at the rs699 (SEQ ID         NO: 14) variable site of AGT, or for the deletion at the         rs1799752 (SEQ ID NO: 12; SEQ ID NO:35) of ACE, then the patient         will likely benefit most from an angiotensin-converting enzyme         (ACE) inhibitor.     -   Patients who are homozygous for cytosine at the rs699 (SEQ ID         NO: 14) will likely benefit most from BOTH ACE inhibition and         angiotensin II (AII) receptor blockade.     -   Patients who are heterozygous for the deletion at the rs1799752         (SEQ ID NO:12; SEQ ID NO:35) and heterozygous for cytosine at         the rs699 (SEQ ID NO: 14) variable site should be administered         other drug classes (e.g., diuretic initially followed by         beta-blockade). Although homozygosity at other sites has a         greater impact on hypertension than heterozygosity at rs1799752         and rs699, this is generally true only if the patient has         combined homozygosity at sites indicating that drug classes         other than vasodilators should be administered.

To ascertain whether a beta-blocker should be administered to a subject the following can be performed.

-   -   Patients homozygous for adenine at the rs3892097 (SEQ ID NO: 10)         variable site of the CYP2D6 gene should initially consider the         use of atenolol and carevdilol as therapy. This is PARTICULARLY         important if the patient is homozygous for cytosine at the         rs1801253 (SEQ ID NO:3) variable site (and expresses arginine at         position 389 of the β₁AR polypeptide), or if the patient is         homozygous for adenine at the rs1801252 variable position (and         expresses serine at position 49 of the β₁AR polypeptide).     -   The rs1042713 (SEQ ID NO:6) and rs1042714 (SEQ ID NO:7) variable         sites are less important of the other polymorphism sites within         the beta-blocker class of drugs and generally indicate patients         who will likely respond to non-selective beta-blockade. Thus,         subjects who are homozygous for guanine at the rs1042713         variable site (and express glycine at about position 16 of the         ADRB2 gene product) as well as subjects who are homozygous for         guanine at the rs1042714 position (and express glutamic acid at         β₂AR position 27) are the most responsive to beta-blocker drugs.     -   If subjects are non-homozygous for polymorphisms in the         beta-blockade class of variants, but are homozygous for cytosine         at the rs1801253 (SEQ ID NO:3) variable site (and expresses         arginine at position 389 of the β₁AR polypeptide), or if         subjects are homozygous for adenine at the rs1801252 variable         position (and expresses serine at position 49 of the β₁AR         polypeptide), the beta-blockade class should be considered a         possible line of therapy if they do not carry functional         mutations within the diuretic and vasodilator classes of drugs.         Polymorphism Detection

The polymorphism present in genes such as ADRB1, ADRB2, cytochrome P450 2D6 (CYP2D6), angiotensin converting enzyme (ACE), angiotensinogen, angiotensin receptors, renin, Na⁺ channels (such as SCNN1A), adducin, sodium (Na⁺) chloride (Cl⁻) co-transporters (such as SLC12A3), and/or WNK1 can be detected by any available procedure. For example, samples of cDNA, genomic DNA, and/or mRNA can be obtained from a subject and the sequences of polymorphic or variant sites can be evaluated by procedures such as nucleic acid amplification (e.g., PCR), reverse transcription, insertion/deletion analysis, primer extension, probe hybridization, SNP analysis, sequencing, restriction fragment length polymorphism, Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight mass spectrometry (MALDI-TOF MS), Sequenom MassArray genotyping, Sanger sequencing, polyacrylamide gel electrophoresis, agarose gel electrophoresis, probe array hybridization analysis, and combinations thereof.

The methods for detecting polymorphisms can therefore involve detecting an alteration in a nucleic acid. As used herein a “nucleic acid” is a DNA or RNA molecule. A nucleic acid can be a segment of genomic DNA (e.g., an entire gene, an intron of a gene, an exon of a gene, a segment that includes regulatory elements, a 5′ non-coding segment, a 3′ non-coding segment, or any combination thereof). The nucleic acid can also be a cDNA (having exons but not introns), an amplicon, an RNA, a primer, or probe.

Probes and/or primers can be used that can hybridize to nucleic acid segments flanking or including of any of SNPs, insertions, deletions, polymorphic, or other variant segments of ADRB1, ADRB2, cytochrome P450 2D6 (CYP2D6), angiotensin converting enzyme (ACE), angiotensinogen, angiotensin receptors, renin, Na⁺ channels (such as SCNN1A), adducin, sodium (Na⁺) chloride (Cl⁻) co-transporters (such as SLC12A3), and/or WNK1 genes. For example, probes and/or primers can be employed that hybridize to nucleic acid segments flanking or including any of the following polymorphisms: rs1801252 (ADRB1), rs1801253 (ADRB1), rs1042713 (ADRB2), rs1042714 (ADRB2), rs3892097 (CYP2D6), rs1799752 (ACE), rs699 (AGT), rs5186 (AGT1R), rs12750834 (renin), rs2228576 (SCNN1A), rs4961 (ADD1), rs1529927 (SLC12A3), rs2107614 (WNK1), or rs1159744 (WNK1). For example, the probes and/or primers can separately hybridize to segments of any of SEQ ID NO:2, 3, 6, 7, 10, 12, 14, 16, 19, 20, 22, 27, 30, 32, 33, 34, as well as to the complementary sequences, amplicons, cDNA, cRNA, and genomic sequences thereof. The probes and/or primers can hybridize to genomic, complementary, amplicon, or cDNA sequences that flank up to 50 nucleotides of any of SEQ ID NO:2, 3, 6, 7, 10, 12, 14, 16, 19, 20, 22, 27, 30, 33, or 34, on either or both of the 5′ and 3′ sides of the polymorphism.

Methods and devices described herein can detect at least two of these polymorphisms, or at least three of these polymorphisms, or at least of four of these polymorphisms, or at least five of these polymorphisms, or at least of six of these polymorphisms, or at least seven of these polymorphisms, or at least of eight of these polymorphisms, or at least nine of these polymorphisms, or at least often of these polymorphisms, or at least eleven of these polymorphisms, or at least of twelve of these polymorphisms, or at least thirteen of these polymorphisms, or at least fourteen of these polymorphisms, or at least fifteen of these polymorphisms or all of these polymorphisms. In some embodiments, the methods and devices described herein detect no other polymorphisms, although such methods and devices can include steps and probes for detecting 1-4 control target nucleic acids. For example, the methods, devices, and kits described herein can detect and evaluate about sixteen polymorphisms.

The probes and primers can be of any convenient length selected by one of skill in the art such as at least 12 nucleotides long, or at least 13 nucleotides long, or at least 14 nucleotides long, or at least 15 nucleotides long, or at least 16 nucleotides long, or at least 17 nucleotides long, or at least 18 nucleotides long, or at least 19 nucleotides long, or at least 20 nucleotides long. In some embodiments, the probes and primers can be less than 150 nucleotides in length, or less than 125 nucleotides in length, or less than 100 nucleotides in length, or less than 75 nucleotides in length, or less than 65 nucleotides in length, or less than 60 nucleotides in length, or less than 55 nucleotides in length, or less than 50 nucleotides in length, or less than 45 nucleotides in length, or less than 40 nucleotides in length.

To detect hybridization, it may be advantageous to employ probes, primers and other nucleic acids in combination with an appropriate detection means. Labels incorporated into primers, incorporated into the amplified product during amplification, or attached to probes that can hybridize to the target, or its amplified product, are useful in the identification of nucleic acid molecules. A number of different labels may be used for this purpose including, but not limited to, fluorophores, chromophores, radiolabels, enzymatic tags, antibodies, chemiluminescence, electroluminescence, and affinity labels. One of skill in the art will recognize that these and other labels can be used with success in this invention.

Examples of affinity labels include, but are not limited to the following: an antibody, an antibody fragment, a receptor protein, a hormone, biotin, dinitrophenyl (DNP), or any polypeptide/protein molecule that binds to an affinity label. Examples of enzyme tags include enzymes such as urease, alkaline phosphatase or peroxidase to mention a few. Colorimetric indicator substrates can be employed to provide a detection means visible to the human eye or spectrophotometrically, to identify specific hybridization with complementary nucleic acid-containing samples. Examples of fluorophores include, but are not limited to, Alexa 350, Alexa 430, AMCA, BODIPY 630/650, BODIPY 650/665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, Cascade Blue, Cy2, Cy3, Cy5, 6-FAM, Fluorescein, HEX, 6-JOE, Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, REG, Rhodamine Green, Rhodamine Red, ROX, TAMRA, TET, Tetramethylrhodamine, and Texas Red.

Means of detecting such labels are well known to those of skill in the art. For example, radiolabels may be detected using photographic film or scintillation counters. In other examples, fluorescent markers may be detected using a photodetector to detect emitted light. In still further examples, enzymatic labels are detected by providing the enzyme with a substrate and detecting the reaction product produced by the action of the enzyme on the substrate, and colorimetric labels are detected by simply visualizing the colored label or by use of spectrometer.

So called “direct labels” are detectable labels that are directly attached to or incorporated into a probe or primer, or to the target (sample) nucleic acid prior to hybridization to a probe that can, for example, be present on a plate, chip, microtiter plate, or microarray. In contrast, so called “indirect labels” are joined to the hybrid duplex after hybridization. In some embodiments, the indirect label is attached to a binding moiety that has been attached to the target nucleic acid prior to the hybridization. Thus, for example, the target nucleic acid may be biotinylated before the hybridization. After hybridization, an avidin-conjugated fluorophore will bind the biotin-bearing hybrid duplexes providing a label that is easily detected. For a detailed review of methods of labeling nucleic acids and detecting labeled hybridized nucleic acids see, for example, Peter C. van der Vliet & Shiv Pillai, eds., Laboratory Techniques in Biochemistry and Molecular Biology (1993).

Probe arrays, assay plates, and gene chip technology provide a means of rapidly screening a large number of nucleic acid samples for their ability to hybridize to a variety of probes immobilized on a solid substrate that is part of the probe array, assay plate, gene chip or microarray. The technology capitalizes on the complementary binding properties of single stranded nucleic acid probe to screen nucleic acid samples by hybridization (Pease et al., Proc. Natl. Acad. Sci. U.S.A. 91: 5022-5026 (1994); U.S. patent to Fodor et al. (1991)). Basically, a nucleic acid probe array or gene chip consists of a solid substrate with an attached array of single-stranded probe molecules. In some embodiments, the probes can fold back on (i.e., hybridize to) themselves to quench a signal from an attached label, but the probes unfold to hybridize to a target nucleic acid, whereupon the signal from the attached label becomes detectable. In other embodiments, the probe can be complementary to the segment of a target nucleic acid but the 3′ end of the probe terminates one nucleotide short of a SNP in the target nucleic acid. The target nucleic acid can be longer than the probe. A signal can be detected upon primer extension of the probe, where the assay mixture contains just one type of labeled nucleotide that can base pair with the variant nucleotide of the SNP. After washing, the presence or absence of the SNP is detectable by incorporation or non-incorporation of the labeled SNP nucleotide into specific probes of the array or plate.

For screening, the chip, plate, or array is contacted with a nucleic acid sample (e.g., genomic DNA, cRNA, cDNA, or amplified copies thereof), which is allowed to hybridize under stringent conditions. The chip, plate, or array is then scanned to determine which targets have hybridized to which probes. The probes are arrayed in known locations so a signal detected at a specific location indicates that its target has hybridized thereto.

Methods for directly synthesizing on or attaching nucleic acid probes to solid substrates are available in the art. See, e.g., U.S. Pat. Nos. 5,837,832 and 5,837,860, both of which are expressly incorporated by reference herein in their entireties. A variety of methods have been utilized to either permanently or removably attach the probes to the substrate. Exemplary methods include: the immobilization of biotinylated nucleic acid molecules to avidin/streptavidin coated supports (Holmstrom, (Anal. Biochem. 209: 278283 (1993)), the direct covalent attachment of short, 5′-phosphorylated primers to chemically modified polystyrene plates (Rasmussen et al., Anal. Biochem. 198: 138-142 (1991)), or the precoating of the polystyrene or glass solid phases with poly-L-Lys or poly L-Lys, Phe, followed by the covalent attachment of either amino- or sulfhydryl-modified oligonucleotides using bifunctional crosslinking reagents (Running et al., BioTechniques 8: 276 277 (1990); Newton, C. R. et al., Acids Res. 21: 1155-1162 (1993)). When immobilized onto a substrate, the probes are typically stabilized and therefore can be used repeatedly.

Hybridization can performed on an immobilized probe that is attached to a solid surface such as silicon, plastic, nitrocellulose, nylon or glass by addition of one or more target molecules. In some embodiments, the target nucleic acid can be attached to a solid surface and the probe can be added to the immobilized target nucleic acids. Numerous substrate and/or matrix materials can be used, including reinforced nitrocellulose membrane, activated quartz, activated glass, polyvinylidene difluoride (PVDF) membranes, polystyrene, polyacrylamide, poly(vinyl chloride), poly(methyl methacrylate), poly(dimethyl siloxane), photopolymers (which contain photoreactive species such as nitrenes, carbenes and ketyl radicals capable of forming covalent links with target molecules), and combinations thereof.

The term “hybridization” includes a reaction in which one or more polynucleotides react to form a complex that is stabilized via hydrogen bonding between the bases of the nucleotide residues. The hydrogen bonding may occur by Watson-Crick base pairing, Hoogstein binding, or in any other sequence-specific manner. The complex may comprise two strands forming a duplex structure, three or more strands forming a multi-stranded complex, a single self-hybridizing strand, or any combination of these. A hybridization reaction may constitute a step in a more extensive process, such as the initiation of a PCR reaction, primer extension, or the enzymatic cleavage of a polynucleotide by a ribozyme.

Hybridization reactions can be performed under conditions of different “stringency”. The stringency of a hybridization reaction includes the difficulty with which any two nucleic acid molecules will hybridize to one another. Under low to medium stringent conditions, nucleic acid molecules at least 60%, 65%, 70%, 75% identical to each other remain hybridized to each other, whereas molecules with lower percent identity cannot remain hybridized. For detection of single base polymorphisms, higher stringency conditions can be used.

A preferred, non-limiting example of highly stringent hybridization conditions include hybridization in 6× sodium chloride/sodium citrate (SSC) at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 50° C., preferably at 55° C., more preferably at 60° C., and even more preferably at 65° C.

When hybridization occurs in an antiparallel configuration between two single-stranded polynucleotides, the reaction is called “annealing” and those polynucleotides are described as “complementary”. A double-stranded polynucleotide can be “complementary” and/or “homologous” to another polynucleotide, if hybridization can occur between one of the strands of the first polynucleotide and the second.

Complementarity” or “homology” (the degree that one polynucleotide is identical or complementary to another) is quantifiable in terms of the proportion of bases in opposing strands that are expected to hydrogen bond with each other, according to generally accepted base-pairing rules.

Detection/Identification of Genetic Variants in Expressed Polypeptides

Genetic variants present in polypeptides such as ADRB1, ADRB2, cytochrome P450 2D6 (CYP2D6), angiotensin converting enzyme (ACE), angiotensinogen, angiotensin receptor, renin, Na⁺ channels (such as SCNN1A), adducin, sodium (Na⁺) chloride (Cl⁻) co-transporters (such as SLC12A3), and/or WNK1 can be detected by use of binding entities such as antibodies. Detection of specific differences in these polypeptides can be used to evaluate which blood pressure mediation is more effective.

Altered polypeptides can be detected in a selected fluid or tissue sample (e.g., cell scrapings, saliva, hair follicle, blood, skin tissue, or any convenient sample of a subject's nucleic acids). Any available methods for detecting polypeptides can be employed. Examples of such methods include immunoassay, Western blotting, enzyme-linked immunosorbant assays (ELISAs), radioimmunoassay, immunocytochemistry, immunohistochemistry, flow cytometry, immunoprecipitation, one- and two-dimensional electrophoresis, mass spectroscopy and/or detection of enzymatic activity.

Altered polypeptides can be detected by binding entities.

Antibodies and other binding entities can be used to detect genetic variants present in ADRB1, ADRB2, cytochrome P450 2D6 (CYP2D6), angiotensin converting enzyme (ACE), angiotensinogen, angiotensin receptors, renin, Na⁺ channels (such as SCNN1A), adducin, sodium (Na⁺) chloride (Cl⁻) co-transporters (such as SLC12A3), and/or WNK1 polypeptides. Such antibodies and binding entities can be prepared by available methods. For example, available amino acid sequences of non-variant and genetic variant ADRB1, ADRB2, CYP2D6, angiotensin converting enzyme (ACE), angiotensinogen, angiotensin receptors, renin, Na⁺ channels (such as SCNN1A), adducin, sodium (Na⁺) chloride (Cl⁻) co-transporters (such as SLC12A3), and/or WNK1, including those illustrated herein, can be used to make antibodies and binding entities. Suitable antibodies may include polyclonal, monoclonal, fragments (such as Fab fragments), single chain antibodies and other forms of specific binding molecules. Briefly, these polypeptide detection assays can include contacting a test sample with an antibody specific to the genetic variant site in the polypeptide, detecting the presence of a complex between the antibody and the polypeptide. In some embodiments, a signal from the polypeptide-antibody complex is detected.

Such antibody-based detection methods can any convenient immuno-detection method such as Western Blot, ELISA, radioimmunoassay, immunocytochemistry, immunohistochemistry, flow cytometry, and immunoprecipitation.

Antibodies can be used to detect or identify the presence of genetic variant forms of ADRB1, ADRB2, CYP2D6, angiotensin converting enzyme (ACE), angiotensinogen, angiotensin receptors, renin, Na⁺ channels (such as SCNN1A), adducin, sodium (Na⁺) chloride (Cl⁻) co-transporters (such as SLC12A3), and/or WNK1 polypeptides in a sample. The antibodies are specific for sites of genetic variations, and exhibit substantially no (or significantly less) binding to similar polypeptides that do not have the same genetic variation(s).

Generally speaking, such antibodies can be employed in any type of immunoassay, so long as the genetic variations in the polypeptides are reliably identified. This includes both the two-site sandwich assay and the single site immunoassay of the non-competitive type, as well as in traditional competitive binding assays.

For example, in a typical forward sandwich assay, unlabeled antibody is immobilized on a solid substrate, e.g., within microtiter plate wells, and the sample to be tested is brought into contact with the bound molecule. After a suitable period of incubation, for a period of time sufficient to allow formation of an antibody-antigen binary complex, a second antibody, labeled with a reporter molecule capable of emitting or inducing a detectable signal, is then added and incubation is continued allowing sufficient time for binding with the antigen at a different site and the formation of a ternary complex of antibody-antigen-labeled antibody. Any unreacted material is washed away, and the presence of the antigen is determined by observation of a signal, which may be quantified by comparison with a control sample containing known amounts of antigen.

Variations on the forward sandwich assay include the simultaneous assay, in which both sample and antibody are added simultaneously to the bound antibody, or a reverse sandwich assay in which the labeled antibody and sample to be tested are first combined, incubated and added to the unlabeled surface bound antibody. These techniques are well known to those skilled in the art, and the possibility of minor variations will be readily apparent. As used herein, “sandwich assay” is intended to encompass all variations on the basic two-site technique.

For the sandwich assays, the only limiting factor is that both antibodies have different binding specificities for the genetic variant polypeptide. Thus, a number of possible combinations are possible. For example, a primary antibody can bind specifically to the variant epitope of one of the variant polypeptides. A secondary antibody can bind to a different site on the genetic variant polypeptide. As a more specific example, in a typical forward sandwich assay, a primary antibody is either covalently or passively bound to a solid support. The solid surface is usually glass or a polymer, the most commonly used polymers being cellulose, polyacrylamide, nylon, polystyrene, polyvinylchloride or polypropylene. The solid supports may be in the form of tubes, beads, discs or microplates, or any other surfaces suitable for conducting an immunoassay.

Conventional antibody binding processes can be employed. Following binding, the solid phase-antibody complex is washed in preparation for the test sample. An aliquot of the test sample is then added to the solid phase complex and incubated at about 25° C. for a period of time sufficient to allow binding of any genetic variant polypeptides present to the antibody. The primary antibody can bind specifically to the site of the genetic variant (e.g., the region of a variant amino acid and/or the structural changes associated therewith), but not to similar polypeptides that have no such genetic variant. After washing off unbound antibodies, the second antibody is then added to the solid phase complex and incubated at 25° C. for an additional period of time sufficient to allow the second antibody to bind to the primary antibody-antigen solid phase complex (e.g., to a different site on the genetic variant polypeptide than is bound by the primary antibody). The second antibody may be linked to a reporter molecule, the visible signal of which is used to indicate the binding of the second antibody to any antigen in the sample.

As used herein, a “reporter molecule” or “label” is a molecule that provides an analytically detectable signal, allowing the detection of antigen-bound antibody. In some embodiments, detection is preferably at least relatively quantifiable, to allow determination of the amount of antigen in the sample, this may be calculated in absolute terms, or may be done in comparison with a standard (or series of standards) containing a known normal level of antigen. The term “label” is used interchangeably with “reporter molecule.”

Many commonly used reporter molecules in this type of assay are either enzymes or fluorophores. In the case of an enzyme immunoassay an enzyme is conjugated to the second antibody, often by means of glutaraldehyde or periodate. As will be readily recognized, however, a wide variety of different conjugation techniques exist, which are well known to the skilled artisan. Commonly used enzymes include horseradish peroxidase, glucose oxidase, beta-galactosidase and alkaline phosphatase, among others. The substrates to be used with the specific enzymes are generally chosen for the production, upon hydrolysis by the corresponding enzyme, of a detectable color change. For example, p-nitrophenyl phosphate is suitable for use with alkaline phosphatase conjugates; for peroxidase conjugates, 1,2-phenylenediamine or toluidine are commonly used. It is also possible to employ fluorogenic substrates, which yield a fluorescent product rather than the chromogenic substrates noted above. In all cases, the enzyme-labeled antibody is added to the first antibody-antigen complex and allowed to bind to the complex, and then the excess reagent is washed away. A solution containing the appropriate substrate is then added to the tertiary complex of antibody-antigen-labeled antibody. The substrate reacts with the enzyme linked to the second antibody, giving a qualitative visual signal, which may be further quantified, usually spectrophotometrically, to give an evaluation of the amount of antigen that is present in the serum sample.

Additionally, fluorescent compounds, such as fluorescein or rhodamine, may be chemically coupled to antibodies without altering their binding capacity. When activated by illumination with light of a particular wavelength, the fluorophore-labeled antibody absorbs the light energy, inducing a state of excitability in the molecule, followed by emission of the light at a characteristic longer wavelength. The emission appears as a characteristic color visually detectable with a light microscope. As in the enzyme immunoassay (EIA), the fluorescent-labeled antibody is allowed to bind to the first antibody-tagged polypeptide complex. After washing the unbound reagent, the remaining ternary complex is then exposed to light of the appropriate wavelength, and the fluorescence observed indicates the presence of the antigen.

Immunofluorescence and EIA techniques are both very well established in the art and are particularly preferred for the present method. However, other reporter molecules, such as radioisotopes, chemiluminescent or bioluminescent molecules may also be employed. It will be readily apparent to the skilled artisan how to vary the procedure to suit the required use.

In another embodiment, the sample to be tested may be used in a single site immunoassay wherein it is adhered to a solid substrate either covalently or non-covalently. An unlabeled antibody is brought into contact with the sample bound on the solid substrate. After a suitable period of incubation, for a period of time sufficient to allow formation of an antibody-antigen binary complex a second antibody, labeled with a reporter molecule capable of inducing a detectable signal, is then added and incubation is continued allowing sufficient time for the formation of a ternary complex of antigen-antibody-labeled antibody. For the single site immunoassay, the second antibody may be a general antibody (i.e., xenogeneic antibody to immunoglobulin, particularly anti-(IgM and IgG) linked to a reporter molecule) that is capable of binding an antibody that is specific for the genetic variant polypeptide of interest.

Kits

Another aspect of the invention is one or more kits for evaluating blood pressure from a test sample provided by, or obtained from, a subject.

The kits can include any reagents, components and instructions useful for testing, assaying, detecting, identifying, and/or determining whether genetic variations are present in ADRB1, ADRB2, CYP2D6, angiotensin converting enzyme (ACE), angiotensinogen, angiotensin receptors, renin, Na⁺ channels (such as SCNN1A), adducin, sodium (Na⁺) chloride (Cl⁻) co-transporters (such as SLC12A3), and/or WNK1 polypeptides or nucleic acids that can be present in the test samples.

The kits can include reagents, components and instructions for detecting, identifying, and/or quantifying such polypeptides or nucleic acids. For example, the kits may include primers, probes, labels, enzymes and/or other components for detecting, and/or identifying genetic variations in such polypeptides or nucleic acids.

In other embodiments, the kits may include one or more antibody preparations that selectively bind to genetic variant ADRB1, ADRB2, CYP2D6, angiotensin converting enzyme (ACE), angiotensinogen, angiotensin receptors, renin, Na⁺ channels (such as SCNN1A), adducin, sodium (Na⁺) chloride (Cl⁻) co-transporters (such as SLC12A3), and/or WNK1 polypeptides, and a detection means for detecting an antibody complex that can form (e.g., a label or reporter molecule that is either bound to an antibody or is capable of binding to the antibody).

One type of kit can include components for obtaining a sample from a subject, and instructions for sample collection. For example, such a sample collection kit can include one or more containers for sample collection such as one or more vials, test tubes, or receptacles. The sample collection containers can include a solution for stabilizing samples placed in the containers. Such a stabilizing solution can include protease inhibitors, nuclease inhibitors, DNase inhibitors, RNase inhibitors, chelators, denaturants, salts, salts, and/or buffers. The sample collection kit can also include components for sample collection such as swabs, droppers, syringes, needles, scalpels, and/or catheters. The instructions can include steps for sample collection, storage of the sample, and submission of the sample.

The kits can include one or more probes and/or primers each capable of specifically binding to a nucleic acid segment of at least 13, 14, 15, 16, 17, 18, 19, 20, or 25 nucleotides. In some embodiments, probes and/or primers are each capable of specifically binding to a nucleic acid segment of 15-30, 15-40, 15-50 nucleotides, or any number of nucleotides between 13-50 nucleotides, in a target DNA or RNA. The probes may be part of an array, microarray, microchip, assay plate, or nanochip.

Alternatively, the probes or primers may be packaged separately and/or individually. In some embodiments, the probes or primers may be detectably labeled. For example, labels can be included on immobilized probes, where the label signals are quenched until hybridization occurs and then, upon hybridization, the label emits a detectable signal. Alternatively, one or more labels can be included in the kit that can bind to a hybridized complex between a probe and a target DNA or RNA.

Additional reagents can be included in the kits. For example, the kits may also contain reagents for detecting or identifying a genetic variant in an ADRB1, ADRB2, CYP2D6, angiotensin converting enzyme (ACE), angiotensinogen, angiotensin receptors, renin, Na⁺ channels (such as SCNN1A), adducin, sodium (Na⁺) chloride (Cl⁻) co-transporters (such as SLC12A3), and/or WNK1 nucleic acid in a test sample. Such reagents can include reagents for isolating, storing and detecting nucleic acids. For example, the kits can include reagents and enzymes for nucleic acid amplification, primer extension, RNA reverse transcription, sequencing, restriction enzyme cleavage, and/or separation of nucleic acids. The kits may also include reagents such as solutions for stabilizing nucleic acids, solutions for purifying nucleic acids, nucleotide triphosphates, buffers, and/or other reagents that can be used in a test tissue sample.

Preservatives and/or antimicrobial agents can be included to stabilize reagents and prevent contamination, such as, for example, paraben, chlorobutanol, phenol sorbic acid, and the like.

It may also be desirable to include agents such as solvents for nucleic acids, reducing agents (e.g., beta-mercaptoethanol), stabilizing reagents (e.g., reagents for inhibiting nucleases, ribonucleases, disrupting tissues, precipitating nucleic acids, and the like).

In further embodiments, the kits can include a computer program product for use in conjunction with a computer system and the methods described herein. A computer program mechanism can be embedded in the computer program product. The computer program product can, for example, be a device with a computer program mechanism encoded thereon, where the computer program mechanism may be loaded into the memory of a computer and cause the computer to carry out at least one step of a method for assessing the malignant/benign status of a test thyroid tissue sample. For example, the device can be a computer readable storage medium, a flash memory, a compact disc (CD), a digital versatile disc, digital video disc, or an article of manufacture that tangibly includes one or more computer programs and memory storage. In some embodiments, the computer program product can be a computer readable storage medium. In such kits, the computer program mechanism can include instructions for determining, detecting, and/or identifying a genetic variant in an ADRB1, ADRB2, CYP2D6, angiotensin converting enzyme (ACE), angiotensinogen, angiotensin receptors, renin, Na⁺ channel (such as SCNN1A), adducin, sodium (Na⁺) chloride (Cl⁻) co-transporter (such as SLC12A3), and/or WNK1 nucleic acid or polypeptide in a test sample.

In other embodiments, the kits can include a system, such as a computer, having a central processing unit and a memory coupled to the central processing unit. The memory may store instructions for determining, detecting, and/or identifying a genetic variant in an ADRB1, ADRB2, CYP2D6, angiotensin converting enzyme (ACE), angiotensinogen, angiotensin receptors, renin, Na⁺ channel (such as SCNN1A), adducin, sodium (Na⁺) chloride (Cl⁻) co-transporter (such as SLC12A3), and/or WNK1 nucleic acid or polypeptide in a test sample. The memory can also store therapeutic options for different genotyping results.

The kits can also include one or more therapeutic agents, for example, any blood pressure medications described herein.

Definitions

Some definitions are provided below; other definitions are provided in the other sections of the applications.

As used herein, “obtaining a test sample” involves removing a sample of tissue from a patient, receiving a sample of tissue from a patient, receiving a patient's tissue sample from a physician, receiving a patient's tissue sample via mail delivery and/or removing a patient's tissue sample from a storage apparatus (e.g., a refrigerator or freezer) or a facility. Thus, obtaining a test sample can involve removal or receipt of the test sample directly from the patient, but obtaining a test sample can also include receipt of a test sample indirectly from a medical worker, from a storage apparatus/facility, from a mail delivery service after transportation from a medical facility, and any combination thereof. The test sample can therefore originate in one location, and be transported to another location where it is received and tested. Any of these activities or combinations of activities involves “obtaining a test sample.”

As used herein a probe refers to a single DNA or RNA molecule (a nucleic acid oligomer) or a collection of nucleic acid molecules (nucleic acid oligomers) where the DNA molecules have at least one segment with a sequence that is complementary to a region of a target nucleic acid. The probe can hybridize with the target nucleic acid under stringent conditions. In some cases, the probe can hybridize with the target nucleic acid under highly stringent conditions. The probe is not identical to naturally available nucleic acids because has additional components such as one or more labels, one or more (engineered) restriction sites, one or more molecular barcodes, one or more tags for identification or retrieval of the probe (e.g., with or without the target hybridized thereto). In some instances the probe is attached to a solid surface such as a chip, an array, a bead, or other surface.

As used herein a primer contains a region that is designed to hybridize to a targeted locus (e.g., a targeted polymorphic locus or a nonpolymorphic locus). The primer and may contain a priming sequence designed to allow PCR amplification. The primer can have at least one segment with a sequence that is complementary to a region of a target nucleic acid. The primer can hybridize with the target nucleic acid under stringent conditions. In some cases, the primer can hybridize with the target nucleic acid under highly stringent conditions. The primer is not identical to naturally available nucleic acids because has additional components such as a molecular barcode, a tag, an engineered restriction site, or a combination thereof. A primer may contain a random region that differs for each individual molecule. The terms “test primer” and “candidate primer” are not meant to be limiting and may refer to any of the primers disclosed herein.

As used herein a “binding entity” is a molecule or molecular complex that can recognize and bind to selected target molecules. Such binding entities can be antibodies or any molecule that has a binding domain for a target molecule.

A number of proteins can serve as protein scaffolds to which binding domains for targets can be attached and thereby form a suitable binding entity. The binding domains bind or interact with the targets of the invention while the protein scaffold merely holds and stabilizes the binding domains so that they can bind. A number of protein scaffolds can be used. For example, phage capsid proteins can be used. See Review in Clackson & Wells, Trends Biotechnol. 12:173-184 (1994). Phage capsid proteins have been used as scaffolds for displaying random peptide sequences, including bovine pancreatic trypsin inhibitor (Roberts et al., PNAS 89:2429-2433 (1992)), human growth hormone (Lowman et al., Biochemistry 30:10832-10838 (1991)), Venturini et al., Protein Peptide Letters 1:70-75 (1994)), and the IgG binding domain of Streptococcus (O'Neil et al., Techniques in Protein Chemistry V (Crabb, L., ed.) pp. 517-524, Academic Press, San Diego (1994)). These scaffolds have displayed a single randomized loop or region that can be modified to include binding domains for selected targets.

Researchers have also used the small 74 amino acid α-amylase inhibitor Tendamistat as a presentation scaffold on the filamentous phage M13. McConnell, S. J & Hoess, R. H., J. Mol. Biol. 250:460-470 (1995). Tendamistat is a β-sheet protein from Streptomyces tendae. It has a number of features that make it an attractive scaffold for binding peptides, including its small size, stability, and the availability of high resolution NMR and X-ray structural data. The overall topology of Tendamistat is similar to that of an immunoglobulin domain, with two β-sheets connected by a series of loops. In contrast to immunoglobulin domains, the β-sheets of Tendamistat are held together with two rather than one disulfide bond, accounting for the considerable stability of the protein. The loops of Tendamistat can serve a similar function to the CDR loops found in immunoglobulins and can be easily randomized by in vitro mutagenesis. Tendamistat is derived from Streptomyces tendae and may be antigenic in humans. Hence, binding entities that employ Tendamistat are preferably employed in vitro.

Fibronectin type III domain has also been used as a protein scaffold to which binding entities can be attached. Fibronectin type III is part of a large subfamily (Fn3 family or s-type Ig family) of the immunoglobulin superfamily. Sequences, vectors and cloning procedures for using such a fibronectin type III domain as a protein scaffold for binding entities (e.g. CDR peptides) are provided, for example, in U.S. Patent Application Publication 20020019517. See also, Bork, P. & Doolittle, R. F. (1992) Proposed acquisition of an animal protein domain by bacteria. Proc. Natl. Acad. Sci. USA 89, 8990-8994; Jones, E. Y. (1993) The immunoglobulin superfamily Curr. Opinion Struct. Biol. 3, 846-852; Bork, P., Hom, L. & Sander, C. (1994) The immunoglobulin fold. Structural classification, sequence patterns and common core. J. Mol. Biol. 242, 309-320; Campbell, I. D. & Spitzfaden, C. (1994) Building proteins with fibronectin type III modules Structure 2, 233-337; Harpez, Y. & Chothia, C. (1994).

The following non-limiting examples further illustrate aspects of the invention.

Example 1: Sample Processing

Each patient is given a collection kit consisting of two buccal swabs and two uniquely barcoded tubes (termed A and B swabs) containing a proprietary lysis buffer consisting of 50 mM Tris pH 8.0, 50 mM EDTA, 25 mM Sucrose, 100 mM NaCl, and 1% SDS. The patient will use the swab to collect buccal cells by scraping the inside of their cheek and place the swab in the provided barcoded tube, one swab for each cheek. Once the swab has been placed into the lysis buffer the cells are no longer viable and therefore samples are now considered to be nucleic acids and safe to be shipped via standard mail. All samples are checked-in. The barcodes of the samples are scanned and their arrival in the laboratory is confirmed.

FIGS. 3A-3B show schematic diagrams illustrating slight variations in sample processing. In general, two samples (Swab A and Swab B) are taken. The Swab A sample is subjected to the process (DNA Extraction through Reporting) unless the Swab A sample fails either the DNA Yield and Purity Assays, Genomic Analysis, or the PCR QA Assay. If such failure occurs, then the other sample (Swab B) is subjected to the process, as illustrated in FIGS. 3A and/or 3B.

The samples are grouped into sets of 91 and assigned positions in 96 sample grids (12×8 grid layout) for DNA extraction. The remaining five positions in each grid can be extraction controls (four negative controls [H₂O] and one non-human positive). The five controls can be assigned random positions in each grid, giving each grid/plate a unique “plate fingerprint.” The randomly assigned controls prevent possible plate swaps or 180° rotations as every plate is now identifiable simply by control positions. All samples are then normalized to a volume of 650 ul by addition of the above mentioned lysis buffer. Additionally, 25 ul of proteinase K (ProK) is added and each sample is incubated in a 55° C. oven for a minimum of 4 hours.

Following such incubation, the samples are extracted using a BioSprint96 (KingFisher96) Robotic workstation with magnetic-particle DNA purification chemistry to isolate genomic DNA (GenomicDNA) from tissue samples. This protocol utilizes the chemistry from the eVoMagDNA Extraction KF96 Kit (Verde Labs, Marietta, Ga.) and is run to specifications provided by the manufacturer.

Following DNA extraction and subsequent desiccation, the DNA is resuspended in HPLC water. Five microliters of each sample is then aliquoted to assay plates for the first pair of QA assays, both a PicoGreen fluorometric quantification and a spectrophotometric purity estimation. The fluorescence and absorbance data is analyzed for all samples in the 96 well plate, including the five controls. The positions of the negative controls is confirmed and accessed for possible plate contamination. The results for the positive control as well as the samples on the plate are analyzed for quality metrics using a systems analysis approach. The outliers are statistically assessed. After the quantification and purity evaluations, QA assay robotic systems are used to transfer the samples into racks of 96 sample septa sealed plates (to ensure there is no evaporative loss) and a fractional volume of each sample is used to create a daughter plate of the samples at a normalized concentration of 5 ng/μl for the PCR QA assays and subsequent genotyping. The creation of the normalized daughter plate serves two purposes. First, it allows the immediate storage of the primary stock of each sample at −80° C. avoiding the need for unnecessary freeze-thaw of samples and the potential contamination risks associated with repeated accessing of the stock. Second, it avoids unnecessary waste of the DNA associated with the use of full concentration stock for the PCR applications (this −80° C. stock DNA can be used at any time or saved for future testing).

Any samples that fail any of the QA assays can re-enter the pipeline and be sorted and re-processed from the B-swab, which is the second tube/swab in the kit sent to the customer mentioned above. By always having a backup sample it is not necessary to go back to the customer to ask for a re-swab. If the quantity and purity are still insufficient then whole genome amplification and/or organic re-extraction can be employed.

Following the passage of the QA thresholds normalized fractions of the samples are transferred to PCR plates for genotyping. Each sample is analyzed using three different methodologies, the Sequenom MassArray genotyping platform, Sanger sequencing using the ABI 3730xl genomic analyzer from Applied Biosystems, and classical PCR and gel sizing to determine insertion/deletion status. The Sequenom MassArray genotyping platform is used to analyze the following SNP sites: rs1042713, rs1042714, rs1159744, rs12750834, rs1801252, rs1801253, rs2107614, rs227869, rs4244285, rs4961, and rs699. Sanger sequencing is used to analyze the following SNPs: rs3892097, rs3758581, rs2228586, and rs5186. Finally classical gel sizing is used to determine the insertion/deletion status of the rs1799752 SNP.

Example 2: Sequenom MassArray Assay Design and Processing

The Sequenom platform is able to perform genotyping as a twelve-plex assay (testing 12 variable sites in one reaction) in a 96 well format using one aliquot of DNA. The AssayDesign software from Sequenom is used to generate both PCR and single base extension primers using the individual rs number of each variable site to create the final assay design. Table 1 shows examples of primers that can be used to detect various single nucleotide polymorphisms.

TABLE 1 Primers for Amplification of Nucleic Acid Variant Segments SNP ID 2^(nd) PCRP SEQ ID 1^(st) PCRP SEQ ID rs1042714 ACGTTGGATGAGACATGACGATGCCCATGC NO: 36 ACGTTGGATGAGCGCCTTCTTGCTGGCAC NO: 37 rs699 ACGTTGGATGCTGTGACAGGATGGAAGACT NO: 38 ACGTTGGATGTGGACGTAGGTGTTGAAAGC NO: 39 rs4961 ACGTTGGATGTGTTCGTCCACACCTTAGTC NO: 40 ACGTTGGATGACAAGATGGCTGAACTCTGG NO: 41 rs12750834 ACGTTGGATGGGAATCCAGGAGAATAGGTC NO: 42 ACGTTGGATGACAGGCTACCTGGCTTTAAC NO: 43 rs1801252 ACGTTGGATGGCCTCGTTGCTGCCTCCCG NO: 44 ACGTTGGATGATCAGCAGACCCATGCCCG NO: 45 rs1801253 ACGTTGGATGAGCCCTGCGCGCGCAGCA NO: 46 ACGTTGGATGTCAACCCCATCATCTACTGC NO: 47 rs227869 ACGTTGGATGCTGACATTGCCAGCTGTATC NO: 48 ACGTTGGATGGTAGTGGCACTGGCATATTC NO: 49 rs2107614 ACGTTGGATGGCAACCATCACAGTACTAAG NO: 50 ACGTTGGATGCACAACTGGAAGAGTTGAGG NO: 51 rs1529927 ACGTTGGATGTGGACCCCATTAACGACATC NO: 52 ACGTTGGATGTCACCTTGGACTCCCACTC NO: 53 rs4244285 ACGTTGGATGCACTTTCCATAAAAGCAAGG NO: 54 ACGTTGGATGGCAATAATTTTCCCACTATC NO: 55 rs1042713 ACGTTGGATGATGAGAGACATGACGATGCC NO: 56 ACGTTGGATGGAACGGCAGCGCCTTCTTG NO: 57 rs1159744 ACGTTGGATGGAAACAGTGACAGCCAAATG NO: 58 ACGTTGGATGGTTTTTCAGTTCCTGAATTTG NO: 59

DNA samples at a concentration of 5 ng/ul undergo a PCR using the above designed PCR primers and the Sequenom iPLEX Gold Reagent kit under the conditions described in Table 2.

TABLE 2 PCR Reaction Mixture Reagent Final Concentration Vol/rxn (uL) Water, HPLC N/A 1.8 10x PCR Buffer with 20 mM MgCl₂  2 mM MgCl₂ 0.5 25 mM MgCl₂  2 mM 0.4 25 mM dNTP Mix 500 uM 0.1 0.5 mM Primer Mix  0.1 uM 1 5 U/uL PCR Enzyme  1 unit 0.2 Volume 4 10 ng/uL DNA  10 ng/rxn 1 Total Volume 5

The PCR reaction cycling conditions can be as illustrated in Table 3.

TABLE 3 PCR Reaction Cycling Cycler Program iPlex- PCR Temp (° C.) Time (min) 95 2:00 95 0:30 Repeat 56 0:30 45 72 1:00 Cycles 72 5:00 4 ∞

Directly following PCR amplification, excess primers and deoxynucleotide triphosphates are removed via a SAP (shrimp alkaline phosphatase) reaction under the conditions described in Table 4.

TABLE 4 PCR Clean-Up Reagent Final Concentration Vol/rxn (uL) Water, HPLC N/A 1.53 SAP Buffer (10x) 0.24x 0.17 5U/uL PCR Enzyme 1 unit 0.2 Volume 2 PCR product 5 Total Volume 7 The Shrimp Alkaline Phosphatase reaction is incubated at 37° C. for 40 min, followed by incubation at 85° C. for 5 min. The samples can be stored at 4° C. indefinitely.

After the SAP reaction is completed the samples can be subjected to single base extension reactions using the primers described in Table 5, and the conditions described in Table 6 and 7.

TABLE 5 Single Base Extension Primers SEQ ID SNP Sequence NO: rs1042714 ACACCTCGTCCCTTT 60 rs699 CTGGCTGCTCCCTGA 61 rs4961 ACTGCTTCCATTCTGCC 62 rs12750834 AGTCTCTGTAAGTGCCC 63 rs1801252 GTGCCTCCCGCCAGCGAA 64 rs1801253 CGCGCGCAGCAGAGCAGT 65 rs227869 AGCTGTATCTGCTCCATTCA 66 rs2107614 TCCTCCAAAAAAAAAGAAAAC 67 rs1529927 GTTACCGACATCCGCATCATTG 68 rs4244285 TAAGTAATTTGTTATGGGTTCC 69 rs1042713 GGAGGGGTCCGGCGCATGGCTTC 70 rs1159744 CAAATGTTAACAGTATAGAAAATTTTA 71

TABLE 6 Single Base Extension Reaction Conditions Reagents Final Concentration Vol/rxn (uL) Water, HPLC N/A 0.619 iPlex Gold Buffer 0.222x 0.200 iPlex Termination Mix 1x 0.200 iPlex Extend Primer Mix varies 0.940 iPlex Enzyme 1x 0.041 Volume 2.000 PCR product 7 Total Volume 9

TABLE 7 Single Base Extension Reaction Cycling conditions Temp (∞C) Time (min) 94 0:30 94 0:05 40 cycles 52 0:05 5 cycles ↓ 80 0:05 ↓ 72 3:00 4 forever

After completion of all of the above reactions, the samples are run through resin based clean-up to remove excess salts according to standard Sequenom protocols. The samples are then spotted onto the Sequenom provided SpectroChip using the Sequenom Nanodispenser according to manufacturer protocols and subsequently processed on the Sequenom MALDI-TOF platform.

A sample results report is provided in Table 7. The two letters for each polymorphism type are for the two alleles present in the subjects, illustrating that the subjects are homozygous for some polymorphisms (e.g., subject GCE0104 is homozygous (G/G) for the variable site in the rs1042713 polymorphism, but subject GCE0120 is heterozygous (GA) for that site).

TABLE 8 Results SNP GCE0120 GCE0104 rs1042713 GA GG rs1042714 GC GG rs1159744 AA AT rs12750834 GA GG rs1529927 GG GG rs1801252 AA AA rs1801253 GG GG rs2107614 CT TT rs227869 AA AG rs4244285 GG GA rs4961 GG GG rs699 CG GG

Example 3: Sanger Sequencing Primer Design and Workflow

All primers for Sanger sequencing were designed using the free, web-based primer design tool Primer3. 500 base pairs of flanking sequence for each SNP (single nucleotide polymorphism) were entered into Primer3. The top primer candidate for each site was chosen and optimized using a buffer panel with varying MgCl concentrations and a temperature gradient to determine the optimal cycling conditions for each primer pair.

TABLE 9 Primers for Sequencing of SNPs Primer Name Sequence SEQ ID Purpose rs3892097_F TTCAGTCCCTCCTGAGCTA NO: 72 SNP rs3892097_R AAGGTGGATGCACAAAGAG NO: 73 SNP rs3758581_F GTGCATCTGTAGCAGTCCTC NO: 74 SNP rs3758581_R CCAAACTGGAATCAACAGAA NO: 75 SNP rs2228586_F GAAGTGGTCTCGTCTAGCAA NO: 76 SNP rs2228586_R CAGAGAGAGAGGTCCCATTT NO: 77 SNP rs5186_F CCACTCAAACCTTTCAACAA NO: 78 SNP rs5186_R TGGACAGAACAATCTGGAAC NO: 79 SNP

The region encompassing the SNP was amplified from sample nucleic acids by PCR using optimized individual cycling conditions for each SNP site. Directly after PCR amplification each sample is cleaned up using a size exclusion micro-filtration plate from Millipore and entered into the Sanger sequencing reaction. Each sample is sequenced in both the forward (3′) and reverse (5′) direction giving double conformation of the allelic state. These forward and reverse sequences from each patient are then aligned to the human reference sequence using the CLC DNA workbench program creating an alignment file from which the allele call is made and added to the final SNP call report.

FIG. 6 illustrates the results from one such alignment.

Example 4: Gel Sizing Primer Design and Workflow

To accurately call the insertion/deletion status for site rs1799752, PCR amplification of sample nucleic acids is performed followed by gel electrophoresis. The PCR primers for this site were also designed and optimized using Primer3 and the above mentioned buffer and temperature gradient. The following primer sequences and PCR conditions were ultimately chosen:

TABLE 10 Primer Sequences for PCR of rs1799752 Insertion/Deletion Primer Name Sequence SEQ ID Purpose rs1799752_F-2 CCCATTTCTCTAGACCTGCT NO: 80 INDEL rs1799752_R-2 GGGATGGTGTCTCGTACATA NO: 81 INDEL

Following PCR amplification, each sample is loaded into its own well of a 2% agarose gel and run at 150 mV for approximately 45 min and stained in a bath of GelRed for 2 hours prior to imaging with UV light. The resulting image is used to score the presence or absence of a 288 bp ALU visually by examining the gel for either the higher molecular weight band (indicating the presence of the 288 bp ALU), the lower molecular weight band (indicating the absence of the 288 bp ALU) or both (indicating a heterozygous state. A sample image of the gel is provided in FIG. 7.

Example 5: Genotyping Reports

Once all tests are performed a report is generated containing all results for each tested patient. One example of a report for subjects GCE0120 and GCE0104, is shown below. The two letters for each polymorphism type are for the two alleles present in the subjects, illustrating that the subjects are homozygous whereas other subjects are heterozygous for the variable site of each polymorphism.

TABLE 11 Results from Analysis of Polymorphisms Polymorphism type GCE0120 GCE0104 Sequenom Results rs1042713 GA GG rs1042714 GC GG rs1159744 CG CG rs12750834 GA GG rs1529927 GG GG rs1801252 AA AA rs1801253 GG GG rs2107614 CT TT rs227869 AA AG rs4244285 GG GA rs4961 GG GG rs699 TT TT Sanger Sequencing Results rs3892097 CC CC rs3758581 GG GG rs3758580 CC CT rs2228586 TT TT rs5186 AC AA Gel Results rs1799752 +/+ +/−

Example 6: Clinical Study Protocol

Clinical Protocol Summary

Study Assessment of the Relationship between Genes that Encode Title: Proteins Important in Blood Pressure Regulation and Blood Pressure Therapy in Patients with Hypertension. Study The Geneticure Pharmacogenetic Testing Kit. The kit Device: contains two buccal swabs with two buffer solution vials to stabilize DNA. These buccal swabs are used for DNA collection which is then extracted for analysis of genes important in high blood pressure. Target The Geneticure Pharmacogenetic Testing Kit is a Indication pharmacogenomic treatment decision support product that for Use: tests for clinically important genetic variants affecting a patient's response to antihypertensive medications. Study This is a post-hoc association study of patients who have Design: been diagnosed with high blood pressure and have been stable on medication treatment for at least 6 months. Study To be enrolled in this study, subjects must meet ALL of the Pop- inclusion criteria and NONE of the exclusion criteria: ulation: Inclusion Criteria   1. Subject is able and willing to provide informed    consent   2. Subject is ≥30 and ≤70 years of age   3. Subject with diagnosis of Hypertension for a    minimum of 1 year   4. Subject has been on the same class/classes of blood    pressure medication for a minimum of 6 months.    Note: A change in dosage, frequency, or specific    medication is acceptable as long as there have been    no changes to the class/classes of medications    prescribed.   5. Subject with a Body Mass Index (BMI) ≥19 and ≤35   6. Subject is currently prescribed and taking one of the    following classes of medications alone or in    combination with each other or a Ca+ channel    blocker.     Diuretics     ACE Inhibitors     Angiotensin Receptor Blocker (ARB)     Beta-blockers Exclusion Criteria   1. Subject has clinically significant kidney disease as    determined by the investigator.   2. Subject has clinically significant cardiac disease as    determined by the investigator.   3. Subject has clinically significant vascular disease as    determined by the investigator.   4. Subject has a diagnosis of secondary hypertension or    is experiencing a complication of pregnancy.   5. Subject is currently prescribed and taking any    additional class of medication(s) for high blood    pressure not included in the list above, with the    exception of a Ca+ channel blocker.   6. Subject has Systolic BP >190 or Diastolic BP >120    documented within the six months prior to visit.   7. Subject has a regular alcohol intake of greater than 21    units per week in the past 6 months   8. Subject has smoked greater than two packs of    cigarettes (total) or equivalent nicotine intake in the    past 6 months.   9. Subject has an anticipated survival less than 12    months.  10. Any other reason that the subject is inappropriate for    study enrollment in the opinion of the Investigator. Primary To assess the relationship between the drug therapy Study class/combination of therapy classes that resulted in the best Objective: blood pressure control for a patient vs. what the Geneticure high blood pressure panel would have predicted. Secondary To assess the clinical time to achieve optimal blood Study pressure treatment. Ob- To assess the number of office visits required to achieve jectives: optimal blood pressure treatment.

1 Introduction

Hypertension (high blood pressure) is one of the most important preventable contributors to disease and death in the United States and represents the most common condition seen in the primary care setting (The sixth report of the Joint National Committee on prevention, detection, evaluation, and treatment of high blood pressure. Arch Intern Med. 157(21):2413-2446 (1997); Chobanian et al. JAMA 289(19):2560-2572 (2003)). According to the American Heart Association, approximately 78 million adults (1 in 3) living in the United States have hypertension with more than 5 million new diagnoses made each year (American Heart Association. Heart and Stroke Statistics—2004 Update. Paper presented at: Dallas: American Heart Association (2004); Roger et al. Circulation. 125(1):e2-e220 (2012)). Of these individuals, 82% are aware they have it, 75% are currently being treated for it, but only 52% have their blood pressure under control. Thus, about 48% of individuals with hypertension do not have adequate blood pressure control. Hypertension is known to lead to myocardial infarction (heart attack), stroke, renal failure, and death if not detected early and treated appropriately. In fact, in 2009, high blood pressure was listed as a primary or contributing cause of death in ˜350,000 of the ˜2.4 million U.S. deaths (14% of all deaths). From 1999-2009 the number of deaths attributable to hypertension increased by 44%.

Refractory (or resistant) hypertension is defined as blood pressure that remains above clinical guideline goals in spite of concurrent use of three antihypertensive agents of different classes (Akpunonu et al., Disease-a-month: DM. October 1996; 42(10):609-722). Critically, refractory hypertension is noted in approximately 25-30% of all individuals being treated for hypertension. Refractory hypertension is a common clinical problem which contributes to the high levels of morbidity and mortality. The inability to gain control of blood pressure in these individuals may be related to the pharmacogenetics of the individual coupled with the specific classes of drugs and/or combination of classes chosen for that individual (Calhoun et al. Circulation 117(25):e510-526 (2008); Johnson & Turner, Curr Opin Mol Ther 7(3):218-225 (2005)). In 2009, the direct and indirect economic burden on the United States health care system associated with hypertension was estimated at $51 billion. With the advent of improved diagnostic techniques, increased rates of health care utilization and screening, and the increasing age of the population, a continual upward trend in this expenditure is expected.

Globally, nearly 1 billion individuals have been diagnosed with hypertension with an estimate of an additional 400 million living with undiagnosed hypertension. Hypertension is the leading cause of premature death and the leading cause of cardiovascular disease worldwide. Similar to the continued upward trend in prevalence as seen in the United States, it is estimated that in 2025 approximately 1.56 billion adults will be living with hypertension. Because nearly two-thirds of the people living with hypertension worldwide reside in developing countries, providing optimal treatment at the lowest cost is critically important.

Unfortunately, despite a significant impulse in the medical community to move towards an “individualized medicine” approach to patient centered treatment, the current clinical treatment strategy is based on a set algorithm which does not take into account individual patient differences. Rather, physicians are guided to choose a drug (one out of many options) in a given class of drugs and use that specific drug as a “first line therapy” (typically initiating with the diuretic class) and titrate that specific drug of choice to therapeutic dosage regardless of efficacy (Chobanian et al. JAMA 289(19):2560-2572 (2003)). It is only after a prolonged course of treatment with that specific class of drug that clinical efficacy is determined (typically three months). At this stage, if clinical guideline goals for blood pressure have not been met, it is often recommended that the patient remain on the “first line therapy” whilst an additional drug from a different class of drugs (typically an Angiotensin converting enzyme inhibitor (ACE inhibitor) or Angiotensin II receptor blocker (ARB)) is added to the pharmacologic regimen. Again, this drug is titrated to recommended therapeutic dosage and another prolonged course of treatment is initiated before clinical efficacy is determined (an additional three months-six months since initiation of treatment). If at this point, clinical guideline goals for blood pressure have not been met, a third drug from a third class of drugs (typically a beta-blocker) is added and the process is repeated (another three months-nine months from initiation of treatment). Further, if clinical guideline goals have continued to be elusive, the diagnosis of refractory hypertension is added and the process is reinitiated with a different combination of drugs, different classes of drugs, different drug options within a given class of drugs, different dosages, or all of the above. Thus, from the time of initial diagnosis and the start of treatment to the point in which blood pressure is adequately controlled may take anywhere from three months to well over one year. This trial-and-error standard of care is clearly not optimal.

The blood pressure panel created by Geneticure has been created to comprehensively assess fourteen common genetic variants in the cardiac, vascular, and renal systems that can improve therapeutic guidance for the clinician based on known functional alterations of the protein through these genetic changes, as well as demonstrated effects of certain drug classes on these various genotypes. Based on this information, a clinician can guide therapy with knowledge specific to their patient, rather than “trial-and-error” based on population data and using drugs with least side effects initially.

1.1 Investigational Device: Geneticure Pharmacogenetic Testing Kit

The Geneticure pharmacogenetic testing kit contains two buccal swabs with two buffer solution vials to stabilize DNA. These buccal swabs are used for DNA collection which is then extracted for analysis of genes important in high blood pressure.

The Geneticure Pharmacogenetic Testing Kit is a pharmacogenomic treatment decision support product that tests for clinically important genetic variants affecting a patient's response to antihypertensive medications.

1.2 Genetic Analysis

Each sample can be analyzed for fourteen common genetic variants using 3 different methodologies, the Sequenom MassArray genotyping platform, Sanger sequencing using the ABI 3730xl genomic analyzer from Applied Biosystems, and classical PCR and gel sizing to determine insertion/deletion status (see, FIGS. 3A-3B).

2 Methodology 2.1 Study Design and Protocol Overview

This is a post-hoc association study of patients who have been diagnosed with high blood pressure and have been stable on medication treatment for at least 6 months. The purpose of this study is to evaluate the relationship between optimal medication therapy (or the therapy that has resulted in the most stable blood pressure for that particular patient) and the predicted optimal medication therapy based on a patient's genetic profile.

Chart reviews for the patient's history of antihypertensive therapy can be coupled with buccal swabs and blood pressure readings collected from eligible patients who have provided informed consent. The swab can be analyzed for fourteen genetic variants that are associated with antihypertensive therapy response (efficacy, side-effects).

2.2 Study Objective

To assess the relationship between the drug therapy class/combination of therapy classes that resulted in the best blood pressure control for a patient vs. what the Geneticure high blood pressure panel would have predicted.

2.3 Secondary Objectives

The secondary objectives are as follows:

-   -   To assess the clinical time to achieve optimal blood pressure         treatment.     -   To assess the number of office visits required to achieve         optimal blood pressure treatment.

3 Investigational Study Center

This study will be conducted at up to 5 study centers within the United States that have adequate resources for trial responsibilities.

4 Study Population

To be enrolled in this study, subjects must meet ALL of the inclusion criteria and NONE of the exclusion criteria:

4.1 Inclusion Criteria

-   -   1. Subject is able and willing to provide informed consent     -   2. Subject is ≤30 and ≤70 years of age     -   3. Subject with diagnosis of Hypertension for a minimum of 1         year     -   4. Subject has been on the same class/classes of blood pressure         medication for a minimum of 6 months. Note: A change in dosage,         frequency, or specific medication is acceptable as long as there         have been no changes to the class/classes of medications         prescribed.     -   5. Subject with a Body Mass Index (BMI) ≥19 and ≤35     -   6. Subject is currently prescribed and taking one of the         following classes of medications alone or in combination with         each other or a Ca⁺ channel blocker.         -   Diuretics         -   ACE Inhibitors         -   Angiotensin Receptor Blocker (ARB)         -   Beta-blockers

4.2 Exclusion Criteria

-   -   1. Subject has clinically significant kidney disease as         determined by the investigator.     -   2. Subject has clinically significant cardiac disease as         determined by the investigator.     -   3. Subject has clinically significant vascular disease as         determined by the investigator.     -   4. Subject has a diagnosis of secondary hypertension or is         experiencing a complication of pregnancy.     -   5. Subject is currently prescribed and taking any additional         class of medication(s) for high blood pressure not included in         the list above, with the exception of a Ca+ channel blocker.     -   6. Subject has Systolic BP >190 or Diastolic BP >120 documented         within the six months prior to visit.     -   7. Subject has a regular alcohol intake of greater than 21 units         per week in the past 6 months     -   8. Subject has smoked greater than two packs of cigarettes         (total) or equivalent nicotine intake in the past 6 months.     -   9. Subject has an anticipated survival less than 12 months.     -   10. Any other reason that the subject is inappropriate for study         enrollment in the opinion of the Investigator.

5 Informed Consent

The investigator will prepare an informed consent form in accordance with this study protocol and all regulatory requirements (21 CFR Part 50) using the template informed consent form provided by the sponsor. The informed consent form must be submitted to the IRB and a copy of the final IRB-approved consent form must be submitted to the Study Management Center prior to the start of the study at that investigational site.

Prior to any study procedures, all subjects must document their consent for study participation and authorization for use and disclosure of health information by signing the IRB-approved Informed Consent Form. As part of the consent process, the subject will have the opportunity to ask questions of, and receive answers from the personnel conducting the study.

The investigator will notify the Study Management Center and the IRB within 5 working days if device use occurs without subject informed consent.

6 Study Assessments and Data Management 6.1 Screening

-   -   Identify Potential Study Subjects. Refer to the Inclusion and         Exclusion Criteria sections of this protocol for a complete list         of eligibility criteria.     -   Obtain Written Informed Consent. Each potential study         participant must be given time to review the IRB-approved         informed consent form, have his/her questions answered to their         satisfaction and sign the form prior to any study procedures         being performed. A subject will be given a copy of the informed         consent form.     -   Review Inclusion/Exclusion Criteria. The investigator and/or         designee will review all criteria to determine if the subject is         eligible for enrollment. Eligibility of all subjects must         personally be confirmed by the Investigator and will be         documented on the CRF.

6.2 Enrollment

-   -   Assign Identification Number to Eligible Subjects. See Protocol         section 6.3.     -   Record Demographics, Antihypertensive Medical History and         current Blood Pressure. Data will be documented in the source         document and recorded on the CRF, including but not limited to         the following:         -   Age         -   Height         -   Weight         -   Race         -   Ethnicity         -   Length of Hypertension diagnosis         -   Previously and currently prescribed antihypertensive             medications         -   Blood pressure measurements

6.3 Specimen Collection

-   -   Collect Buccal Specimen.     -   Using the collection kit consisting of two buccal swabs and two         uniquely barcoded tubes the investigator or designee will remove         the first buccal brush and scrape the brush end across a         Subject's right cheek repeatedly (for five seconds). The         investigator/designee will place the brush end over the open         buffer vial and press the opposite end of the swab stick to         release the brush into the buffer and then close the vial. The         process can be repeated on the left cheek. Each of the right and         left cheek vial numbers must be recorded on the CRF and         accountability log as right (R), or left (L).     -   Adverse Event Recording     -   Perform Product Accountability

6.4 Subject Numbering

Subjects meeting the criteria for enrollment (and their specimens) can be identified by unique numbers that can be assigned sequentially by order of enrollment. The pre-assigned investigational site number can be prefixed to the identification number and separated by a hyphen (e.g., site 01 would number their subjects sequentially as 01-001, 01-002, 01-003, etc.). Throughout the descriptions within the protocol the A swab will be referring to the swab that has originated from the right cheek, while the B swab will be that that has originated from the left cheek. To further clarify. Subject 01-001 can be given two barcoded tubes. These barcode numbers can be recorded for each patient. These can also be recorded as originating from the right cheek (A) or left cheek (B).

At no time should any study paperwork or specimens be marked with the subject's name or any other traceable identifier except for the informed consent form, which is signed by the subject and kept at the site. At no time should the original (signed) or a copy of this form be collected by the Sponsor or its representative.

6.5 Subject Completion and Withdrawal

Once subjects undergo the sampling procedure, their study participation is complete. There are no follow-up visits. Subjects will be instructed to notify the Investigator if they experience any symptoms or complications from the sampling procedure.

Subjects are free to withdraw consent and discontinue participation in the study at any time. A subject's participation in the study may be discontinued at any time at the discretion of the Investigator or Geneticure. The following may be justifiable reasons for the Investigator or Geneticure to remove a subject from the study:

-   -   The subject was erroneously included in the study or was found         to have an exclusion criterion.     -   The subject was uncooperative.     -   The subject experienced an AE/SAE during the sample collection         procedure that is considered intolerable by the subject or         Investigator.

To the extent possible, safety data will be collected on subjects who discontinue participation in the study due to safety reasons.

The following may be justifiable reasons for the Investigator or Geneticure to remove a specimen from the study:

-   -   Sample is determined to be of poor or inadequate quality for         analysis (e.g., contamination, insufficient material for         analysis).     -   The sample was erroneously included in the study.     -   The specimen was not collected or processed per protocol         procedures.

6.6 Concomitant Medications/Treatment/Procedures

This study protocol does not require change to any existing treatments or those prescribed during the course of the study by the Investigator or any other provider whom the subject sees for any medical reason. Outside of eligibility screening, there are no clinical evaluations as part of this study.

6.7 Data Management

The Investigator is responsible to ensure the accuracy, completeness, and timeliness of reported data.

All data will be sent to Geneticure who will enter it into the study database using a secure, protected Excel spreadsheet. The database will be validated prior to use in the study. All required data will be recorded on CRFs or paper facsimiles. Data collected within the CRFs will be supported by source documents as appropriate and may be updated to reflect the latest observations on the subjects participating in the study. Corrections to the source documentation can be made in a manner that does not obscure the original entry and will be dated and initialed by the Investigator or assigned designee. It is important for data entry to occur in a timely manner, therefore, data collected on source documents should be transferred into CRFs as soon as possible following study visits.

Study subject data can be reviewed at the investigational site by monitors at regular intervals throughout the study. Information on the CRFs can be compared to information originally recorded on source documents related to the study (i.e. professional notes, study-specific worksheets, etc.)

7 Genomic Core Laboratory

The subjects' cheek vials will be sent to the Geneticure processing center. The vials will then be batched and sent to the Genomic Core laboratory for DNA extraction and genetic analysis. Following analysis, results will be sent to Geneticure for statistical analysis and DNA will be destroyed.

A protocol for the extraction and analysis will be followed to ensure consistency and objectivity.

8 Adverse Events

The procedures outlined in this protocol do not involve significant risk to subject safety. Subjects will be provided the investigator's contact information and will be instructed to notify the investigator of any adverse events they experience during or secondary to the sample collection procedures.

8.1 Definitions 8.1.1 Adverse Event

-   -   For the purposes of this study, an adverse event is defined as         any undesirable/unusual medical experience that occurs to a         subject in conjunction with the use of the product, whether or         not considered product related, including (but not limited to)         those events that result from the use as stipulated in the         protocol.     -   The following adverse events will not be collected in this         study:         -   Adverse events which, in the opinion of the Investigator,             are unrelated to the swab collection procedure, but rather             related to the subject's underlying medical conditions or             status         -   Adverse events that may be related to the sample collection             procedure but result only in local, mild and transient             discomforts.     -   The Investigator is responsible for documenting all Adverse         Events on the Adverse Event CRF, except for those events noted         above.

8.1.2 Serious Adverse Event

A Serious adverse event is an adverse event that:

-   -   led to death,     -   led to serious deterioration in the health of a subject that         -   resulted in a life-threatening illness or injury,         -   resulted in permanent impairment of a body structure or body             function,         -   required inpatient hospitalization or prolongation of             existing hospitalization,         -   resulted in medical or surgical intervention to prevent             permanent impairment to a body structure or a body function.     -   led to fetal distress, fetal death or a congenital anomaly or         birth defect.

8.2 Event Reporting

Any AE, or SAE experienced by a subject after signing the informed consent until twenty-four (24) hours following study completion or termination will be recorded in the progress notes and on the CRF. The Investigator and/or designee will continue to monitor the subject with additional assessments until the AE is considered resolved, stabilized, or is lost to follow up.

A full description of an adverse event, including the nature, date and time of onset and resolution, determination of seriousness, frequency, severity, treatment, outcome, and relationship to the study will be recorded on the Adverse Event CRF.

SAEs must be reported to RCRI within 48 hours of the Investigator's first knowledge of the event.

9 Statistical Methods

Following is a summary of the Statistical Analysis Plan for the study. The following objectives have been prospectively defined; however, due to the nature of these data, additional analyses may be conducted or additional subsets may be identified that are not listed in this protocol.

9.1 Sample Size

-   -   Up to 300 subjects may be enrolled at each site. The minimum         number of subjects for meaningful statistical analysis is 100         subjects.

9.2 Data Analysis

-   -   All data will be coded for statistical analysis (i.e. drug         classes and genotypes will be coded numerically). All data will         be analyzed with SPSS v.20. Normality of the data will be         assessed using Levene's test prior to statistical analysis and         any correction for non-normal data distribution will be used.         Descriptive statistics will be computed (average time for blood         pressure control, average number of visits to the clinician for         blood pressure control, age, height, weight, BMI, etc.).     -   Data will be initially analysed following the collection of         samples/data from 100 subjects. This will allow for direction         for power calculations/etc. for future statistical analysis.         Although some of the genes have been analysed individually, no         mean or standard deviation data exists to allow for a true a         priori power calculation. Data will be analysed again after two         months or following 300 subjects for which data has been         collected. Statistical tests will be corrected for the number of         tests run (preservation of alpha).     -   Ordinary least squares regression via univariate modelling will         be used to estimate the magnitude of linearity between drug         class that yielded the best blood pressure control and genetic         profile of the subject. Multiple regression analysis will be         performed to determine the impact of confounding variables         (height, weight, age, race) on blood pressure control. For all         statistical analyses an alpha level of 0.05 will be used to         determine statistical significance.

9.3 Other Statistical Considerations

Justification of Pooling Data Across Centers

There is no need to keep the data from different centers separate for data collection. Primary reasons for not pooling blood pressure data from different centers could include different races (which we are collecting as a demographic and analyzing as a co-variate in a multiple-regression) and different cultures (i.e. southern vs. northern habits of diet, exercise, etc.). The study will take race, height, weight, age into account as co-variates in a multiple regression model, but will not be powered to take into account possible geographic influences on the data.

Missing Data

All patients with available data will be included in the analyses of primary and secondary objectives. Because some of the data was not recorded as part of a prospective protocol, an unknown amount of data will be permanently missing. No patients will be contacted to retrieve missing data, and no sensitivity analyses will be performed on missing data.

10 Risk Analysis 10.1 Device Description

The procedures outlined in this protocol do not involve significant risk to subject safety. Subjects will be provided the investigator's contact information and will be instructed to notify the investigator of any adverse events they experience during or secondary to the specimen collection procedures.

The collection kit includes a small, soft, brush for cheek swabbing and a buffer solution in a small vial, one of each for each cheek, two in total. Once the swab has been placed into the lysis buffer the cells are no longer viable and therefore samples are now considered to be nucleic acids and safe to be shipped via standard mail.

11 Study Materials 11.1 Handling and Storage

-   -   The Investigator must ensure that the investigational product is         stored in a controlled location with limited access.

11.2 Product Accountability

-   -   The investigator is responsible for investigational product         accountability, reconciliation and record maintenance. The         investigator must maintain investigational product         accountability records throughout the course of the study.     -   Upon completion or termination of the study, all unused product,         together with a copy of the product accountability form will be         returned to Geneticure or its representative.     -   All supplies are to be used only for this protocol and not for         any other purpose.

12 Study Administration 12.1 Subject Confidentiality

All information and data sent to Geneticure, and/or its designees concerning subjects and their participation in this study are considered confidential by Geneticure and it designees (subcontractors or contract research organization). Only authorized Geneticure personnel or approved contracted agents of Geneticure will have access to some portions of these confidential files and will act in accordance with applicable regulations as required by HIPAA. The IRBs and FDA also have the right to inspect and copy all records pertinent to this study. All data used in the reporting of the study will eliminate identifiable reference to the subjects.

12.2 Investigational Center Qualification

Investigational Center qualification visits or phone calls will be conducted by the Study Management Center prior to acceptance of the site into this study. The site qualification visit will be scheduled to include time with the Principal Investigator and other study personnel as applicable. Areas of discussion include a review of personnel training, investigator qualifications, adequacy of potential subject pool, FDA-regulated study experience, this study's specific requirements for procedures, and a review of staffing availability and appropriateness. A written report of the qualification visit will be drafted by the Study Management Center.

12.3 Site Training

Study-specific training of study personnel is the responsibility of the Sponsor or Study Management Center and the Principal Investigator. Study training will occur before the first device use. To ensure protocol and regulatory compliance as well as accurate data collection, site training will include a detailed review of the protocol, CRF completion, study specific procedures, monitoring logistics, and regulatory requirements.

12.4 Investigator Responsibilities

The investigator is responsible for ensuring that the study is conducted according to the investigational plan and all applicable FDA regulations, including reporting and record-keeping requirements, and controlling the devices undergoing investigation and HIPAA. In addition, the principal investigator is responsible for ensuring that informed consent is obtained from each subject prior to participating in the study, as well as protecting the rights, safety and welfare of participating subjects. Specific responsibilities are listed in this investigational plan.

Records and reports must remain on file at the investigational site for a minimum of two years after the later of either the completion/termination of the investigational study or the date it is determined the records are no longer required to support submissions to regulatory authorities. They may be discarded only upon approval from Geneticure. The Principal Investigator must contact Geneticure before destroying any records and reports pertaining to the study to ensure that they no longer need to be retained. In addition, Geneticure must be contacted if the investigator plans to leave the investigational site to ensure that arrangements for a new investigator or records transfer are made prior to investigator departure.

12.4.1 Records

-   -   Records to be maintained by the investigator in the designated         investigational center's study file include:         -   Investigational plan and all amendments         -   Signed Financial Disclosure         -   IRB approval letter including consent and HIPAA             authorization form(s)         -   IRB Membership list or Letter of Assurance         -   All correspondence relating to the study between the site             and Geneticure, and the Study Management Center         -   CVs and professional licenses for all investigators         -   Site personnel signature and responsibility list         -   Clinical monitor sign-in log         -   Blank set of each version of CRFs         -   Subject Screening/Enrollment log         -   Investigational device accountability log including: date,             quantity, lot numbers of all devices, identification of all             persons the device was used on and final disposition.

The following records are maintained for each subject enrolled in the study:

-   -   Signed Consent Form and Authorization for the Use and Disclosure         of Health Information     -   Compete, accurate and current CRFs and DCFs     -   Adverse event reports and any supporting documentation     -   Protocol deviations     -   Complete medical records, including procedure reports, lab         reports, professional notes, etc.

Geneticure reserves the right to secure data clarification and additional medical documentation on subjects enrolled in this study at any time.

13 Abbreviations

-   -   AE=Adverse Event CRF=Case Report Form     -   DCF=Data Clarification Form FDA=Food and Drug Administration     -   HIPAA=Health Insurance Portability and Accountability Act of         1996     -   IRB/IEC=Institutional Review Board/Independent Ethics Committee     -   ITT=Intent-to-Treat PP=Per Protocol     -   SAE=Serious Adverse Event     -   UADE=Unanticipated Adverse Device Effect

Example 7: Results and Summary of Phase I Clinical Study

Introduction:

For this phase-I research study 14 genes within the Geneticure blood pressure (BP) panel were assessed as they relate to time to BP control and absolute BP values in 99 patients with hypertension. The study design utilized a post-hoc patient chart review carried out by two clinical sites through the direction of RCRI (a third-party clinical research firm) exploring genes important in drug metabolism, renal Na⁺ handling, vascular function, and cardiac output (all of which can result differences in BP and response to BP therapy). Although the primary aim was BP control in response to therapy relative to genetic data, the time on average, it takes patients to achieve BP control without consideration of genetic information was also determined.

In summary, the study demonstrated that the genes in the Geneticure panel were predictive of time to BP control in patients with hypertension. In addition, there was an effect of several of the genes being predictive of BP taken within the clinic at the time of the research study. In addition, mechanistic data was gathered for the genes that encode the alpha subunit of the epithelial Na⁺ channel (SCNN1A, rs#2228576) and found that SCNN1A was predictive of urinary Na⁺ concentration and mean arterial BP.

Methods:

The BP history for patients was collected and the current BP levels were measured in patients with controlled hypertension. DNA was collected using a buccal swab and analyzed the genes within the Geneticure panel. The study sought to determine if patients with “functional” genotypes of proteins important in certain drug classes responded better if they were taking the drug that would inhibit the activity of that protein. As an example, the beta-1 adrenergic receptor (ADRB1) is important in heart rate control and patients who are on a beta-blocker can demonstrate a drop in BP because of inhibition of this protein. Therefore, one would hypothesize that if a patient with a functional protein of the ADRB1 is put on a beta-blocker early, they will demonstrate better BP control (because of a greater drop in heart rate and, therefore BP). This was assessed according to 14 genes and 3 major classes of BP drugs (diuretic, vasodilator, beta-blocker) and one drug metabolizing enzyme (CYP2D6).

Results: Subject Characteristics

Demographics (n=99)

Variable mean ± SEM Age (yrs) 58 ± 0.8 Sex (% female) 46 Diabetes (% with) 28 ± 4   Weight (kg) 86 ± 1.4 Height (cm) 169 ± 1   BMI (kg/m²) 29.9 ± 0.4   Results: Blood Pressure Data (n=99)

Variable Mean ± SEM Initial SBP (mmHg) 151 ± 2 Initial DBP (mmHg)  91 ± 1 Initial MAP (mmHg) 111 ± 1 Lowest SBP in past two years (mmHg) 115 ± 1 Lowest DBP in past two years (mmHg)  72 ± 1 Current SBP (mmHg) 134 ± 2 Current DBP (mmHg)  82 ± 1 Current MAP (mmHg)  99 ± 1 Time to BP control (months)  22 ± 10 Clinic Visits in the Past two years for HTN  3.6 ± 0.3 Results: Current Blood Pressure Therapy Information Drug Class Usage (n=99)

Variable mean ± SEM Number of Classes of Drugs for HTN  1.8 ± 0.08 Diuretic (% taking) 42 ± 5 ACE Inhibitor (% taking) 62 ± 5 ARB (% taking) 27 ± 5 B-Blocker (% taking) 33 ± 5 Ca⁺ Channel Blocker (% taking) 16 ± 4 These data describe the number of different drug classes that the patients were taking. In addition, we assessed the percent of subjects who were on drugs within the vasodilator class (ACE-inhibitor and ARB), the cardiac class (B-blocker Ca⁺ channel blocker), and the renal class (diuretic). Time to Control According to Drug Class (n=99)

Months For Control Clinic Visits/2 Years On the Not on the On the Not on the Drug Class Drug Class Drug Class Drug Class Drug Class Diuretic 39.5 ± 20.4 7.9 ± 4.2 4.5 ± 0.6 3.0 ± 0.4* ACE Inhibitor 22.2 ± 11.4 22.5 ± 16.4 3.1 ± 0.4 4.5 ± 0.6* Antiotensin 32.8 ± 23.1 17.1 ± 9.1  3.9 ± 0.6 3.5 ± 0.4  Receptor Blocker B-Blocker 24.5 ± 16.9 21.2 ± 12.0 4.9 ± 0.7 3.1 ± 0.4* Ca+ Channel 9.9 ± 4.5 25.0 ± 11.7 5.1 ± 0.7 3.3 ± 0.4  Blocker *p < 0.05 compared to those patients who were on the class of drugs

These data describe the time it took for BP control according to which class of drugs the patient was taking. While there are no significant differences in months taken for BP control according to drug class, there was an effect of number of clinic visits (specific to hypertension) within the past 2 years according to drug class. Patients using beta-blockade and diuretic therapy to control their BP had fewer clinic visits, when compared to those patients not on these therapies. Patients on an ACE-inhibitor had significantly more clinic visits per year, when compared to patients not on this therapy.

Blood Pressure Control According to Genotypes (n=86)

1. Genes Important in Renal Na⁺ Handling (and Those that are Differentially Responsive to Diuretic Therapy).

WNK1 (RS# 1159744) On Target Therapy (Diuretic) No Yes Genotype GG C•containing GG C•containing n 26 24 19 15 Systolic 133.7 ± 3.2  133.5 ± 2.3  137.8 ± 4.1  132.1 ± 5.2  Blood Pressure (mmHg) Diastolic 79.7 ± 2.3 84.6 ± 2.0 88.7 ± 2.7* 79.5 ± 3.7 Blood Pressure Mean 97.7 ± 2.4 100.9 ± 1.81 105.1 ± 2.7*  97.1 ± 3.6 Arterial Blood Pressure (mm Hg) Months to  3.6 ± 1.4  4.8 ± 2.6 8.2 ± 5.6 16.5 ± 6.2 BP Control *P < 0.05 compared to same genotype not on target therapy.

SLC12A3 (RS# 1529927) On Target Therapy (Diuretic) No Yes Genotype GG C•containing GG C•containing n 45 6 33 2 Systolic Blood 134.2 ± 2.31 136.8 ± 5.9 136.4 ± 3.4 128.0 ± 8   Pressure (mmHg) Diastolic Blood 81.8 ± 1.7  86.5 ± 2.3  85.9 ± 2.5 75.0 ± 5.0 Pressure (mm Hg) Mean Arterial 99.3 ± 1.7 103.3 ± 3.1 102.7 ± 2.4 92.3 ± 6.0 Blood Pressure (mm Hg) Months to BP  2.5 ± 0.7 17.7 ± 7   10.5 ± 3.9 42 Control

WNK1 (RS# 2107614) On Target Therapy No Yes Genotype GG C•containing G C•containing n 9 41 7 27 Systolic Blood 130.3 ± 5.6  134.4 ± 2.1  147.7 ± 7.9 132.1 ± 3.3 Pressure (mmHg) Diastolic Diastolic Blood 80.4 ± 4.7 82.4 ± 1.6 84.6 ± 4   84.7 ± 2.8 Pressure (mm Hg) Mean Arterial 97.1 ± 4.6 99.7 ± 1.6  106 ± 4.4 100.5 ± 2.6 Blood Pressure (mm Hg) Months to BP 10.5 ± 7.2  2.6 ± 0.9  10.5 ± 9.5  13.1 ± 4.8 Control

Alpha Adducin (RS# 4961) On Target Therapy No Yes Genotype GG T•containing GG T•containing n 40 11 24 11 Systolic Blood 134.2 ± 2.3 135.0 ± 5.3  135.3 ± 3.9 137.4 ± 5.9 Pressure (mmHg) Diastolic Blood  83.1 ± 1.8 79.7 ± 3.3  85.8 ± 2.9  84.2 ± 4.3 Pressure (mm Hg) Mean Arterial 100.2 ± 1.8 98.2 ± 3.6 102.3 ± 2.7 101.9 ± 4.5 Blood Pressure (mm Hg) Months to BP  4.4 ± 1.6  3.6 ± 15.1  10.5 ± 5.2  15.1 ± 6.7 Control

Of the four genes explored in the clinical study RS#1159744 (the gene that encodes cytoplasmic serine-threonine kinase that is expressed in the kidney, WNK-1) was most predictive of response to therapy. Patients with the C genotype of WNK-1 had the best response to therapy demonstrating 8 mmHg lower DBP, when compared to patients with this genotype who were not on diuretic therapy. Subjects who were homozygous for G for this gene actually had a lower blood pressure if they were not on a diuretic, indicating that they may be benefiting from alternate therapy. Although just a trend (due to small sample size of the minor allele) the C polymorphism of SLC12A3 also may be predictive of response to diuretic therapy with patients demonstrating an 11 mmHg drop in DBP with therapy, compared to the G polymorphism which demonstrated a small increase in DBP with therapy.

In addition to this clinical BP data 24-hour urinary and resting BP data were gathered according to genetic variation of the alpha sub-unit of the epithelial Na⁺ channel (SCNN1A, RS#2228576). It was found that subjects homozygous for the T variant of SCNN1A demonstrated more Na⁺ excretion from the kidneys and they also demonstrated lower mean arterial blood pressure, when compared to genotype groups containing the A variant (See FIGS. 8 and 9).

2. Genes Important in Cardiac Function (and Those that May Respond Differentially to Beta-Blocker Therapy).

The beta-1 adrenergic receptor (ADRB1) is important in controlling heart rate and cardiac contractility.

Beta-1 Adrenergic Receptor 49 (RS# 1801252) On Target Therapy No Yes (Beta-Blocker) Thr•con- Thr•con- Genotype Ser/Ser taining Ser/Ser taining n 1 55 2 28 Systolic Blood 137 136.4 ± 2.3 136 ± 6  132.4 ± 3.2  Pressure (mmHg) Diastolic Diastolic Blood 86  85.5 ± 1.7 75 ± 3 80.3 ± 2.1* Pressure (mm Hg) Mean Arterial 103 102.4 ± 1.7 95 ± 0 97.7 ± 2.1  Blood Pressure (mm Hg) Months to BP N/A  7.0 ± 2.4 N/A 9.4 ± 3.8 Control *P < 0.05 compared to same genotype not on target therapy.

Beta-1 Adrenergic Receptor 389 (RS# 1801253) On Target Therapy (Beta- Blocker) No Yes Genotype GG C•containing GG C•containing n 56 0 30 0 Systolic 136.4 ± 2.3 132.6 ± 3.0  Blood Pressure (mmHg) Diastolic Diastolic  85.5 ± 1.7 80.0 ± 2.0* Blood Pressure (mm Hg) Mean 102.4 ± 1.7 97.5 ± 1.98 Arterial Blood Pressure (mm Hg) Months to  7.0 ± 2.3 8.8 ± 3.5 BP Control *P < 0.05 compared to same genotype not on target therapy.

These data indicate a differential BP response to beta-blocker therapy according to genetic variation at position 49 of the ADRB1. Specifically, the inventors found that subjects with the Ser genotype at position 49 of ADRB1 benefit from beta-blocker therapy with an average drop in DBP of 11 mmHg, compared with a drop of 5 mmHg with Thr at this position. Therefore, although patients with the Thr polymorphism also demonstrated a drop in BP with beta-blocker therapy, the effect was most pronounced in patients with the Ser polymorphism.

The beta-2 adrenergic receptor (ADRB2) is important in cardiac contractility, which controls stroke volume, and can influence BP through differences in cardiac output.

Beta-2 Adrenergic Receptor 16 (RS# 1042713) On Target Therapy No Yes (B-Blocker) Gly-con- Gly-con- Genotype Arg/Arg taining Arg/Arg taining n 5 51 3 27 Systolic Blood 143.6 ± 5.9  135.7 ± 2.4 129 ± 5  133.3 ± 3    Pressure (mmHg) Diastolic Blood 84.0 ± 7.4  85.6 ± 1.8 80.3 ± 6.6 79.9 ± 2.1* Pressure (mmHg) Mean Arterial 104.0 ± 6.82 102.3 ± 1.8 96.6 ± 5.9 97.6 ± 2.1  Blood Pressure (mmHg) Months to BP  7 ± 5    7 ± 2.6  4.3 ± 3.8 9.9 ± 4.3 Control *P < 0.05 compared to same genotype not on target therapy

Beta-2 Adrenergic Receptor 27 (RS# 1042714) On Target Therapy No Yes (B-Blocker) Glu-con- Glu-con- Genotype Gln/Gln taining Gln/Gln taining n 6 48 4 24 Systolic Blood 142.0 ± 6.9 134.4 ± 2.2 125.5 ± 5.9 132.7 ± 3.6  Pressure (mmHg) Diastolic Blood  82.5 ± 2.7  85.1 ± 1.8  82.5 ± 2.7 79.5 ± 2.4 Pressure (mmHg) Mean Arterial 102.3 ± 4.1 101.5 ± 1.8 102.3 ± 4.1 97.2 ± 2.3 Blood Pressure (mmHg) Months to  8.5 ± 7.8    7 ± 2.8  5.3 ± 2.3  9.6 ± 4.4 BP Control

These data demonstrate a generally favorable response to beta-blocker therapy with both genotype groups. However, the Gly16 genotype demonstrated a statistically significant difference in BP control if the patients were on a beta-blocker (drop in DBP of 5 mmHg), when compared to patients with the Arg16 genotype. Generally, there is a similar pattern for a greater drop in BP with subjects who have the most functional gene that encodes the ADRB2 (Gly at position 16 and Glu at position 27). There is strong linkage disequilibrium between these two sites (amino acids 16 and 27), so the similar response between the sites is expected.

Observations on Genetic Variation of Cytochrome P450 2D6 (CYP2D6), which is Important in Drug Metabolism, Especially of Particular Beta-Blockers.

CYP 2D6 (RS#) On Target Therapy (B-Blocker) No Yes Genotype CC T-Containing CC T-Containing n 35 22 23 7 Systolic 140.6 ± 2.7 128.5 ± 3.1  133.4 ± 3.6  130.0 ± 5.7  Blood Pressure (mmHg) Diastolic  86.0 ± 2.4 83.2 ± 2.4 79.2 ± 2.1* 82.6 ± 5.3 Blood Pressure (mmHg) Mean 104.2 ± 2.3 98.3 ± 2.4 97.3 ± 2.2* 98.4 ± 4.8 Arterial Blood Pressure (mmHg) Months to  7.8 ± 2.9 5.08 ± 3.7 6.25 ± 3.2   16.5 ± 10.8 BP Control *P < 0.05 compared to same genotype not on target therapy

These data demonstrate that the CC homozygous group of CYP2D6 demonstrates the greatest response to beta-blocker therapy, when compared to the CT and TT groups. Patients with the CC polymorphism had demonstrated a 6 mmHg lower DBP and a 7 mmHd lower MAP when on beta-blocker therapy, compared to this genotype not on beta-blocker therapy. In contrast, patients in the T-containing group (those with the CT and TT genotypes) did not respond to beta-blocker therapy.

3. Genes Important in Vascular Function (and Those that May Respond Differentially to Vasodilator Therapy).

The following are observations on the genetic variation of the angiotensin gene (encoding a precursor to angiotensin-II, a potent vasoconstrictor, which is converted via angiotensin converting enzyme, ACE) and the responses to various therapies.

Angiotensin (RS# 699) On Target Therapy (Angiotensin Receptor Blocker) No Yes Genotype CC T-containing CC T-containing n 15 50  4 17 Systolic Blood 135.3 ± 2.6 135.1+2.6 135.0 ± 7.4  135.1 ± 4.5 Pressure (mmHg) Diastolic Blood  89.5 ± 2.9  82.6 ± 1.7 77.5 ± 7.8  82.7 ± 3.4 Pressure (mmHg) Mean Arterial 104.7 ± 2.6 100.1 ± 1.7 96.6 ± 7.5 100.2 ± 3.3 Blood Pressure (mmHg) Months to  2.7 ± 1.3  6.7 ± 2.7 12  12.4 ± 4.8 BP Control

Angiotensin (RS# 699) On Target Therapy (ACE- Inhibitor) No Yes Genotype CC T-containing CC T-containing n 6 24 13 43 Systolic 131.7 ± 5.3  133.7 ± 3.5  136.8 ± 2.7  135.8 ± 2.9 Blood Pressure (mmHg) Diastolic 78.5 ± 5.0 80.4 ± 2.1 90.8 ± 3.2*  83.9 ± 2.0 Blood Pressure (mmHg) Mean 96.2 ± 4.7   98.2± 106.2 ± 2.8*  101.1 ± 2.0 Arterial Blood Pressure (mmHg) Months to  6.7 ± 3.1  7.7 ± 3.2 2.3 ± 1.7  9.9 ± 3.8 BP Control *P < 0.05 compared to same genotype not on target therapy

All Receptor Type-1 (RS# 5186) On Target Therapy (Angiotensin Receptor Blocker) No Yes Genotype AA C-containing AA C-containing n 39 28 9 11 Systolic Blood 137.3 ± 2.6 131.0 ± 3.1  136.3 ± 4.6 134.1 ± 6.5  Pressure (mmHg) Diastolic Blood  85.4 ± 2.1 80.9 ± 2.0  87.6 ± 3.9 77.9 ± 4.5 Pressure (mmHg) Mean Arterial 102.7 ± 2.0 97.6 ± 2.1 103.8 ± 2.9 96.6 ± 4.9 Blood Pressure (mmHg) Months to  7.3 ± 2.9  3.8 ± 2.5  13.4 ± 7.8 11.9 ± 5.7 BP Control

These data indicate that patients homozygous for the C genotype of angiotensin may benefit from an angiotensin receptor blocker (ARB). When on an ARB, patients with the CC genotype demonstrated a 12 mmHg lower DBP when compared to patients with this genotype who were not on this therapy. In contrast, patients in the T-containing group (those with the CT or TT genotypes) did not show a response to ARB therapy. Furthermore, inhibition of ACE (which converts angiotensin-I to angiotensin-II) results in higher BP levels, possibly due to a “build-up” of angiotensin. Therefore, these data indicate that patients homozygous for C should be given an angiotensin receptor blocker with an ACE inhibitor.

Angiotensin Converting Enzyme (ACE) Genotype and ACE-Inhibition

ACE (RS# 1799752) On Target Therapy (ACE- No Yes Inhibition) Del• Del• Genotype Ins/Ins Containing Ins/Ins Containing n 3 27 12 47 Systolic 142.7 ± 11.1 132.3 ± 3.1  129.5 ± 3.5  137.1 ± 2.6 Blood Pressure (mmHg) Diastolic 79.6 ± 6.1 80.0 ± 2.1 80.4 ± 4.4  85.7 ± 1.9* Blood Pressure (mm Hg) Mean 100.7 ± 7.7  97.4 ± 2.1 96.8 ± 3.8 102.8 ± 1.8 Arterial Blood Pressure (mm Hg) Months to  4.7 ± 3.7 8.0 ± 3   7.4 ± 5.8  7.3 ± 3.0 BP Control *P < 0.05 compared to same genotype not on target therapy.

These data indicate that those with the Insertion polymorphism (Ins) of the ACE gene will respond best to ACE-inhibition. Patients with the Del polymorphism actually demonstrated higher DBP with ACE-inhibition, when compared to this patient group not on ACE-inhibitors.

Observations on Renin Genotype and Angiotensin Receptor Blocker

Renin is a precursor to angiotensin and, therefore, patients with a functional genotype of renin may benefit from Angiotensin Receptor Blocker (ARB) therapy because a more functional genotype can lead to greater angiotensin levels which can result in high BP.

Renin (RS# 12750834) On Target Therapy (Angiotensin Receptor Blocker) No Yes Genotype G/G A•containing G/G A•containing n 48 17 14 7 Systolic 134.8 ± 2.3 135.9 ± 4.4 136.4 ± 4.6 132.2 ± 7.3  Blood Pressure (mmHg) Diastolic  83.8 ± 1.7  85.1 ± 2.9  82.0 ± 2.9 81.0 ± 7.4 Blood Pressure (mm Hg) Mean 100.8 ± 1.7 102.1 ± 2.9 100.2 ± 3.2 98.1 ± 6.6 Arterial Blood Pressure (mm Hg) Months to  4.75 ± 1.9  8.0 ± 5.8  15.5 ± 5.3*  1.3 ± 0.7 BP Control *P < 0.05 compared to same genotype not on target therapy.

These data indicate that the functional genotype of renin (A) may benefit from ARB therapy. Specifically, the BP response to therapy was not significant, however, the response to therapy time was pronounced. Patients who have the functional genotype of renin (the AG and AA genotype groups) demonstrate a much shorter time to BP control, when compared to patients within this group who do not take this therapy. In contrast, patients in the GG group demonstrate a longer time to control if they take this therapy, possibly due to greater response to another class of drugs.

REFERENCES

-   1. Kearney P M, Whelton M, Reynolds K, Muntner P, Whelton P K, He J.     Global burden of hypertension: Analysis of worldwide data. Lancet     2005; 365:217-223. -   2. Brodde O E. The functional importance of beta 1 and beta 2     adrenoceptors in the human heart. Am J Cardiol 1988; 62:24C-29C. -   3. Snyder E M, Wong E C, Foxx-Lupo W T, Wheatley C M, Cassuto N A,     Patanwala A E. Effects of an inhaled beta2-agonist on cardiovascular     function and sympathetic activity in healthy subjects.     Pharmacotherapy 2011; 31:748-756. -   4. Johnson J A, Turner S T. Hypertension pharmacogenomics: Current     status and future directions. Curr Opin Mol Ther 2005; 7:218-225. -   5. La Rosee K, Huntgeburth M, Rosenkranz S, Bohm M, Schnabel P. The     arg389gly beta1-adrenoceptor gene polymorphism determines     contractile response to catecholamines. Pharmacogenetics 2004;     14:711-716. -   6. Liu J, Liu Z-Q, Tan Z-R, Chen X-P, Wang L-S, Zhou G, Zhou H-H.     Gly389arg polymorphism of [beta]1-adrenergic receptor is associated     with the cardiovascular response to metoprolol[ast]. Clin Pharmacol     Ther 2003; 74:372-379. -   7. Snyder E M, Beck K C, Dietz N M, Eisenach J H, Joyner M J, Turner     S T, Johnson B D. Arg16gly polymorphism of the {beta}2-adrenergic     receptor is associated with differences in cardiovascular function     at rest and during exercise in humans. J Physiol 2006; 571:121-130. -   8. Snyder E M, Hulsebus M L, Turner S T, Joyner M J, Johnson B D.     Genotype related differences in beta2 adrenergic receptor density     and cardiac function. Med Sci Sports Exerc 2006; 38:882-886. -   9. Snyder E M, Johnson B D, Joyner M J. Genetics of beta2-adrenergic     receptors and the cardiopulmonary response to exercise. Exerc Sport     Sci Rev 2008; 36:98-105. -   10. Snyder E M, Joyner M J, Turner S T, Johnson B D. Blood pressure     variation in healthy humans: A possible interaction with beta-2     adrenergic receptor genotype and renal epithelial sodium channels.     Med Hypotheses 2005; 65:296-299. -   11. Snyder E M, Turner S T, Joyner M J, Eisenach J H, Johnson B D.     The arg16gly polymorphism of the {beta}2-adrenergic receptor and the     natriuretic response to rapid saline infusion in humans. J Physiol     2006; 574:947-954. -   12. Ulgen M S, Ozturk O, Alan S, Kayrak M, Turan Y, Tekes S,     Toprak N. The relationship between angiotensin-converting enzyme     (insertion/deletion) gene polymorphism and left ventricular     remodeling in acute myocardial infarction. Coron Artery Dis 2007;     18:153-157. -   13. McNamara D M, Holubkov R, Postava L, Janosko K, MacGowan G A,     Mathier M, Murali S, Feldman A M, London B. Pharmacogenetic     interactions between angiotensin-converting enzyme inhibitor therapy     and the angiotensin-converting enzyme deletion polymorphism in     patients with congestive heart failure. J Am Coll Cardiol 2004;     44:2019-2026. -   14. Pilati M, Cicoira M, Zanolla L, Nicoletti I, Muraglia S,     Zardini P. The role of angiotensin-converting enzyme polymorphism in     congestive heart failure. Congest Heart Fail 2004; 10:87-93; quiz     94-85. -   15. Pilbrow A P, Palmer B R, Frampton C M, Yandle T G, Troughton R     W, Campbell E, Skelton L, Lainchbury J G, Richards A M, Cameron V A.     Angiotensinogen m235t and t174m gene polymorphisms in combination     doubles the risk of mortality in heart failure. Hypertension 2007;     49:322-327. -   16. Tang W, Devereux R B, Rao D C, Oberman A, Hopkins P N, Kitzman D     W, Arnett D K. Associations between angiotensinogen gene variants     and left ventricular mass and function in the hypergen study. Am     Heart J 2002; 143:854-860. -   17. Miller J A, Thai K, Scholey J W. Angiotensin ii type 1 receptor     gene polymorphism predicts response to losartan and angiotensin ii.     Kidney Int 1999; 56:2173-2180. -   18. Baudin B. Angiotensin ii receptor polymorphisms in hypertension.     Pharmacogenomic considerations. Pharmacogenomics 2002; 3:65-73. -   19. Vangjeli C, Clarke N, Quinn U, Dicker P, Tighe O, Ho C, O'Brien     E, Stanton A V. Confirmation that the renin gene distal enhancer     polymorphism ren-5312c/t is associated with increased blood     pressure. Circulation Cardiovascular genetics 2010; 3:53-59. -   20. Meisler M H, Barrow L L, Canessa C M, Rossier B C. Scnn1, an     epithelial cell sodium channel gene in the conserved linkage group     on mouse chromosome 6 and human chromosome 12. Genomics 1994;     24:185-186. -   21. Jin H S, Hong K W, Lim J E, Hwang S Y, Lee S H, Shin C, Park H     K, Oh B. Genetic variations in the sodium balance-regulating genes     enac, nedd41, ndfip2 and usp2 influence blood pressure and     hypertension. Kidney Blood Press Res 2010; 33:15-23. -   22. Pratt J H. Central role for enac in development of hypertension.     J Am Soc Nephrol 2005; 16:3154-3159. -   23. Zhang L N, Ji L D, Fei L J, Yuan F, Zhang Y M, Xu J. Association     between polymorphisms of alpha-adducin gene and essential     hypertension in chinese population. BioMed research international     2013; 2013:451094. -   24. Psaty B M, Smith N L, Heckbert S R, Vos H L, Lemaitre R N,     Reiner A P, Siscovick D S, Bis J, Lumley T, Longstreth W T, Jr.,     Rosendaal F R. Diuretic therapy, the alpha-adducin gene variant, and     the risk of myocardial infarction or stroke in persons with treated     hypertension. JAMA 2002; 287:1680-1689. -   25. Turner S T, Schwartz G L, Chapman A B, Boerwinkle E. Wnk1 kinase     polymorphism and blood pressure response to a thiazide diuretic.     Hypertension 2005; 46:758-765. -   26. The sixth report of the Joint National Committee on prevention,     detection, evaluation, and treatment of high blood pressure. Arch     Intern Med. Nov. 24, 1997; 157(21):2413-2446. -   27. Chobanian A V, Bakris G L, Black H R, et al. The Seventh Report     of the Joint National Committee on Prevention, Detection,     Evaluation, and Treatment of High Blood Pressure: the JNC 7 report.     Jama. May 21, 2003; 289(19):2560-2572. -   29. American Heart Association. Heart and Stroke Statistics—2004     Update. Paper presented at: Dallas: American Heart Association 2004. -   30. Roger V L, Go A S, Lloyd-Jones D M, et al. Heart disease and     stroke statistics—2012 update: a report from the American Heart     Association. Circulation. Jan. 3, 2012; 125(1):e2-e220. -   31. Akpunonu B E, Mulrow P J, Hoffman E A. Secondary hypertension:     evaluation and treatment. Disease-a-month: DM. October 1996;     42(10):609-722. -   32. Calhoun D A, Jones D, Textor S, et al. Resistant hypertension:     diagnosis, evaluation, and treatment: a scientific statement from     the American Heart Association Professional Education Committee of     the Council for High Blood Pressure Research. Circulation. Jun. 24,     2008; 117(25):e510-526. -   33. Johnson J A, Turner S T. Hypertension pharmacogenomics: current     status and future directions. Curr Opin Mol Ther. June 2005;     7(3):218-225.

All patents and publications referenced or mentioned herein are indicative of the levels of skill of those skilled in the art to which the invention pertains, and each such referenced patent or publication is hereby specifically incorporated by reference to the same extent as if it had been incorporated by reference in its entirety individually or set forth herein in its entirety. Applicants reserve the right to physically incorporate into this specification any and all materials and information from any such cited patents or publications.

The specific methods, devices, and kits described herein are representative of preferred embodiments and are exemplary and not intended as limitations on the scope of the invention. Other objects, aspects, and embodiments will occur to those skilled in the art upon consideration of this specification, and are encompassed within the spirit of the invention as defined by the scope of the claims. It will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. The invention illustratively described herein suitably may be practiced in the absence of any element or elements, or limitation or limitations, which is not specifically disclosed herein as essential. The methods and processes illustratively described herein suitably may be practiced in differing orders of steps, and the methods and processes are not necessarily restricted to the orders of steps indicated herein or in the claims.

As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to “a nucleic acid” or “a polypeptide” includes a plurality of such nucleic acids or polypeptides (for example, a solution of nucleic acids or polypeptides or a series of nucleic acid or polypeptide preparations), and so forth. Under no circumstances may the patent be interpreted to be limited to the specific examples or embodiments or methods specifically disclosed herein. Under no circumstances may the patent be interpreted to be limited by any statement made by any Examiner or any other official or employee of the Patent and Trademark Office unless such statement is specifically and without qualification or reservation expressly adopted in a responsive writing by Applicants.

The terms and expressions that have been employed are used as terms of description and not of limitation, and there is no intent in the use of such terms and expressions to exclude any equivalent of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention as claimed. Thus, it will be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims and statements of the invention. 

What is claimed:
 1. A method for treating an individual patient having hypertension, the method comprising administering to the individual patient a first line therapy antihypertensive drug selected from the group consisting of a diuretic drug, a vasodilator drug and a beta blocker drug; wherein the diuretic drug is selected from the group consisting of a thiazide diuretic drug, a loop diuretic drug and an amiloride potassium sparing diuretic drug and the vasodilator drug is selected from the group consisting of an angiotensin II receptor antagonist (blocker) drug and an ACE inhibitor drug; the method of administering comprising the steps of: determining the individual patient's genotype for each of fourteen single nucleotide polymorphisms: rs1159744; rs2107614; rs4961; rs1529927; rs3892097; rs1801253; rs1801252; rs12750834; rs5186; rs1799752; rs699; rs2228576; rs1042714; rs1042713; and, administering to the individual patient the first line therapy antihypertensive drug according one of the following procedures a, b, c, d, e, f or g: a) administering a diuretic drug as the first line therapy antihypertensive drug to the patient without administering the betablocker drug, and the vasodilator drug wherein the patient's genotype has been determined to be homozygous for cytosine at the variable position of rs1159744 or rs2107614; b) administering a thiazide or thiazide-like diuretic drug as the first line therapy antihypertensive drug to the patient without administering the betablocker drug, and the vasodilator drug wherein the individual patient's genotype has been determined to be homozygous for thymine at the variable position of rs4961, or homozygous for thymine at the variable position of rs1529927; c) administering the beta-blocker drug as the first line therapy antihypertensive drug to the patient without administering the diuretic drug, and the vasodilator drug wherein the individual patient's genotype has been determined to be homozygous for:
 1. adenine at the variable position of rs3892097;
 2. cytosine at the variable position of rs1801253;
 3. adenine at the variable position of rs1801252;
 4. guanine at the variable position of rs1042714; or
 5. guanine at the variable position of rs1042713; And the patient's genotype is not homozygous for:
 1. cytosine at the variable position of rs1159744;
 2. cytosine at the variable position of rs2107614;
 3. thymine at the variable position of rs4961; and
 4. thymine at the variable position of rs1529927; d) administering the angiotensin II receptor blocker vasodilator drug as the first line therapy antihypertensive drug to the patient without administering the diuretic drug, the beta-blocker drug and the ACE inhibitor vasodilator drug, wherein the patient's genotype has been determined to be homozygous for:
 1. cytosine at the variable position of rs12750834; or
 2. cytosine at the variable position of rs5186; And the patient's genotype is not homozygous for:
 1. cytosine at the variable position of rs1159744;
 2. cytosine at the variable position of rs2107614;
 3. thymine at the variable position of rs4961; and
 4. thymine at the variable position of rs1529927; e) administering the ACE inhibitor vasodilator drug as the first line therapy antihypertensive drug to the patient without administering the angiotensin II receptor blocker vasodilator drug, the diuretic drug and the beta-blocker drug wherein the individual patient's genotype has been determined to be homozygous for:
 1. a deletion in rs1799752; or
 2. cytosine at the variable position of rs699; And the patient's genotype is not homozygous for:
 1. cytosine at the variable position of rs1159744;
 2. cytosine at the variable position of rs2107614;
 3. thymine at the variable position of rs4961; and
 4. thymine at the variable position of rs1529927; f) administering an amiloride potassium sparing diuretic drug as the first line therapy antihypertensive drug to the patient without administering the vasodilator drug, the loop diuretic drug, the thiazide diuretic drug, and the beta-blocker drug, wherein the individual patient's genotype has been determined to be homozygous for: adenine at the variable position of rs2228576; And the patient's genotype is not homozygous for:
 1. cytosine at the variable position of rs1159744;
 2. cytosine at the variable position of rs2107614;
 3. thymine at the variable position of rs4961; and
 4. thymine at the variable position of rs1529927; or g) administering the loop diuretic drug as the first line therapy antihypertensive drug to the patient when the individual patient's genotype is not homozygous for the polymorphic variants of procedures c1-5, d1-2 and e1-2, and is not homozygous for rs2228576 with variable position adenine, rs1529927 with variable position thymine, rs4961 with variable position thymine, rs1159744 with variable position cytosine and rs2107614 with variable position cytosine.
 2. A method according to claim 1 further comprising obtaining a sample of the patient's fluid or tissue and analyzing the sample to determine the fourteen single nucleotide polymorphisms.
 3. A method according to claim 2 wherein the fourteen single nucleotide polymorphisms are determined sequentially or simultaneously.
 4. A method according to claim 3 wherein the single nucleotide polymorphisms are determined simultaneously.
 5. A method according to claim 1 wherein the loop diuretic drug is selected from furosemide, butmetamide, ethacrynic acid or torsemide; the potassium sparing diuretic drug is selected from amiloride, eplerenone, spironolactone or triamterene; and the thiazide-like diuretic drug is selected from chlorothiazide, hydrochlorothiazide, indapamide, metolazone, chlorthalidone, hydroflumthiazide, methylchlothiazide, cyclothaizide, bendroflumethaizide, cyclopenthiazide, polythiazide, benzthiazide, ethiazide, penflutizide or isosorbide.
 6. A method according to claim 1 wherein the ACE inhibitor is selected from enalapril, lisinopril, captopril alacipril, benazapril, cilazapril, delapril, fosinopril, perindopril, quinapril, ramipril, moveltipril, spirapril, ceronapril, imidapril, temocapril, trandolopril, utilbapril, zofenopril, CV5975, EMD 56855, libenzapril, zalicipril, HOE065, MDL 27088, AB47, DU 1777, MDL 27467A, or Y23785.
 7. A method according to claim 1 wherein the angiotensin II receptor antagonist is selected from losartan, valsartan, candesartan, irbesartan, olmesartan, or any combination thereof.
 8. A method according to claim 1 wherein the beta blocker is propranolol, atenolol, bisoprolol, carvedilol, metoprolol, or metoprolol tartrate.
 9. A method according to claim 1 wherein the individual patient to whom is administered a first line therapy antihypertensive drug according to one of procedures a-g exhibits an improved blood pressure response to the first line therapy antihypertensive drug relative to the response the individual patient would exhibit as a result of administration of an antihypertensive drug which is different than the drug administered according to claim
 1. 